Factor viii chimeric and hybrid polypeptides, and methods of use thereof

ABSTRACT

The present invention provides methods of administering Factor VIII (processed FVIII, single chain FVIII, or a combination thereof); methods of administering chimeric and hybrid polypeptides comprising Factor VIII; chimeric and hybrid polypeptides comprising Factor VIII; polynucleotides encoding such chimeric and hybrid polypeptides; cells comprising such polynucleotides; and methods of producing such chimeric and hybrid polypeptides using such cells

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: sequencelisting_ascii.txt; Size: ______ bytes; and Dateof Creation: ______) filed with the application is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of therapeutics forhemostatic disorders.

BACKGROUND ART

Hemophilia A is an X-linked bleeding disorder caused by mutations and/ordeletions in the factor VIII (FVIII) gene resulting in a deficiency ofFVIII activity (Peyvandi, F. et al. Haemophilia 12:82-89 (2006). Thedisease is characterized by spontaneous hemorrhage and excessivebleeding after trauma. Over time, the repeated bleeding into muscles andjoints, which often begins in early childhood, results in hemophilicarthropathy and irreversible joint damage. This damage is progressiveand can lead to severely limited mobility of joints, muscle atrophy andchronic pain (Rodriguez-Merchan, E. C., Semin. Thromb. Hemost. 29:87-96(2003), which is herein incorporated by reference in its entirety).

The A2 domain is necessary for the procoagulant activity of the factorVIII molecule. Studies show that porcine factor VIII has six-foldgreater procoagulant activity than human factor VIII (Lollar, P., and E.T. Parker, J. Biol. Chem. 266:12481-12486 (1991)), and that thedifference in coagulant activity between human and porcine factor VIIIappears to be based on a difference in amino acid sequence between oneor more residues in the human and porcine A2 domains (Lollar, P., etal., J. Biol. Chem. 267:23652-23657 (1992)), incorporated herein byreference in its entirety.

Treatment of hemophilia A is by replacement therapy targetingrestoration of FVIII activity to 1 to 5% of normal levels to preventspontaneous bleeding (Mannucci, P. M., et al., N. Engl. J. Med.344:1773-1779 (2001), which is herein incorporated by reference in itsentirety). There are plasma-derived and recombinant FVIII productsavailable to treat bleeding episodes on-demand or to prevent bleedingepisodes from occurring by treating prophylactically. Based on the shorthalf-life of these products, however, e.g., 8-12 hours, treatmentregimens require the administration of frequent intravenous injections.Such frequent administration is painful and inconvenient.

Reduced mortality, prevention of joint damage and improved quality oflife have been important achievements due to the development ofplasma-derived and recombinant FVIII. Prolonged protection from bleedingwould represent another key advancement in the treatment of hemophilia Apatients. However, to date, no products that allow for prolongedhemostatic protection have been developed. Therefore, there remains aneed for improved methods of treating hemophilia due to factor VIIIdeficiency that are more tolerable, longer lasting, and more effectivethan current therapies.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of administering Factor VIII;methods of administering chimeric polypeptides comprising Factor VIIIand hybrids of such chimeric polypeptides; chimeric polypeptidescomprising Factor VIII and hybrids of such chimeric polypeptides;polynucleotides encoding such chimeric and hybrid polypeptides; cellscomprising such polynucleotides; and methods of producing such chimericand hybrid polypeptides using such cells.

The present invention provides a method of administering Factor VIII toa subject in need thereof, comprising administering to the subject atherapeutic dose of a chimeric Factor VIII polypeptide, e.g., a chimericFactor VIII-Fc polypeptide, at a dosing interval at least about one andone-half times longer than the dosing interval required for anequivalent dose of said Factor VIII without the non-Factor VIII portion(a polypeptide consisting of said Factor VIII portion), e.g., withoutthe Fc portion.

The dosing interval may be at least about one and one-half to six timeslonger, one and one-half to five times longer, one and one-half to fourtimes longer, one and one-half to three times longer, or one andone-half to two times longer, than the dosing interval required for anequivalent dose of said Factor VIII without the non-Factor VIII portion(a polypeptide consisting of said Factor VIII portion), e.g., the Fcportion. The dosing interval may be at least about one and one-half,two, two and one-half, three, three and one-half, four, four andone-half, five, five and one-half or six times longer than the dosinginterval required for an equivalent dose of said Factor VIII without thenon-Factor VIII portion (a polypeptide consisting of said Factor VIIIportion), e.g., the Fc portion. The dosing interval may be about everyfive, six, seven, eight, nine, ten, eleven, twelve, thirteen, orfourteen days or longer.

The dosing interval may be at least about one and one-half to 5, one andone-half, 2, 3, 4, or 5 days or longer.

The present invention also provides a method of administering FactorVIII to a subject in need thereof, comprising administering to thesubject a therapeutic dose of a chimeric Factor VIII polypeptide, e.g.,a chimeric Factor VIII-Fc polypeptide, to obtain an area under theplasma concentration versus time curve (AUC) at least about one andone-quarter times greater than the AUC obtained by an equivalent dose ofsaid Factor VIII without the non-Factor VIII portion (a polypeptideconsisting of said Factor VIII portion), e.g., without the Fc portion.

The present invention also provides a method of administering FactorVIII to a subject in need thereof, comprising administering to thesubject a therapeutic dose of a polypeptide comprising a Factor VIII andan Fc at a dosing interval of about every three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, or fourteen days or longer.

The methods of the invention may be practiced on a subject in need ofprophylactic treatment or on-demand treatment.

On-demand treatment includes treatment for a bleeding episode,hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage intomuscles, oral hemorrhage, trauma, trauma capitis (head trauma),gastrointestinal bleeding, intracranial hemorrhage, intra-abdominalhemorrhage, intrathoracic hemorrhage, bone fracture, central nervoussystem bleeding, bleeding in the retropharyngeal space, bleeding in theretroperitoneal space, or bleeding in the illiopsoas sheath. The subjectmay be in need of surgical prophylaxis, peri-operative management, ortreatment for surgery. Such surgeries include, e.g., minor surgery,major surgery, tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, total knee replacement, craniotomy, osteosynthesis, traumasurgery, intracranial surgery, intra-abdominal surgery, intrathoracicsurgery, or joint replacement surgery.

For on-demand treatment, the dosing interval of said chimericpolypeptide is about once every 24-36, 24-48, 24-72, 24-96, 24-120,24-144, 24-168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72hours or longer.

The therapeutic doses that may be used in the methods of the inventionare about 10 to about 100 IU/kg, more specifically, about 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU/kg, and morespecifically, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 IU/kg.

The therapeutic doses that may be used in the methods of the inventionare about 10 to about 150 IU/kg, more specifically, about 100-110,110-120, 120-130, 130-140, 140-150 IU/kg, and more specifically, about110, 115, 120, 125, 130, 135, 140, 145, or 150 IU/kg.

The subject in the methods of the invention is a human subject. Thedetermination of dosing interval and AUC may be carried out in a singlesubject or in a population of subjects.

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) is ahuman Factor VIII. The Factor VIII (or Factor VIII portion of a chimericpolypeptide) may have a full or partial deletion of the B domain.

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) maybe at least 90% or 95% identical to a Factor VIII amino acid sequenceshown in Table 2 without a signal sequence (amino acids 20 to 1457 ofSEQ ID NO:2 or amino acids 4 to 2351 of SEQ ID NO:6). The Factor VIII(or Factor VIII portion of a chimeric polypeptide) may be identical to aFactor VIII amino acid sequence shown in Table 2 without a signalsequence (amino acids 20 to 1457 of SEQ ID NO:2 or amino acids 20 to2351 of SEQ ID NO:6).

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) maybe at least 90% or 95% identical to a Factor VIII amino acid sequenceshown in Table 2 with a signal sequence (amino acids 1 to 1457 of SEQ IDNO:2 or amino acids 1 to 2351 of SEQ ID NO:6). The Factor VIII (orFactor VIII portion of a chimeric polypeptide) may be identical to aFactor VIII amino acid sequence shown in Table 2 with a signal sequence(amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ IDNO:6).

The Fc portion (or Fc portion of a chimeric polypeptide) may be at least90% or 95% identical to the Fc amino acid sequence shown in Table 2(amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 ofSEQ ID NO:6). The Fc portion (or Fc portion of a chimeric polypeptide)may be identical to the Fc amino acid sequence shown in Table 2 (aminoacids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ IDNO:6).

The chimeric polypeptide may comprise a sequence at least 90% or 95%identical to the Factor VIII and Fc amino acid sequence shown in Table2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO:2)or at least 90% or 95% identical to the Factor VIII and Fc amino acidsequence shown in Table 2A(i) with a signal sequence (amino acids 1 to1684 of SEQ ID NO:2). The chimeric polypeptide may comprise a sequenceidentical to the Factor VIII and Fc amino acid sequence shown in Table2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO:2)or identical to the Factor VIII and Fc amino acid sequence shown inTable 2A(i) with a signal sequence (amino acids 1 to 1684 of SEQ IDNO:2).

The chimeric polypeptide may be in the form of a hybrid comprising asecond polypeptide in association with said chimeric polypeptide,wherein said second polypeptide comprises or consists essentially of anFc.

The second polypeptide may comprise or consist essentially of a sequenceat least 90% or 95% identical to the amino acid sequence shown in Table2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ ID NO:4)or at least 90% or 95% identical to the amino acid sequence shown inTable 2A(ii) with a signal sequence (amino acids 1 to 247 of SEQ IDNO:4). The second polypeptide may comprise or consist essentially of asequence identical to the amino acid sequence shown in Table 2A(ii)without a signal sequence (amino acids 21 to 247 of SEQ ID NO:4) oridentical to the amino acid sequence shown in Table 2A(ii) with a signalsequence (amino acids 1 to 247 of SEQ ID NO:4).

The chimeric polypeptide or hybrid may be administered as part of apharmaceutical composition comprising at least one excipient.

The invention also provides the above-described chimeric and hybridpolypeptides themselves, polynucleotides encoding them, a cultured humanembryonic cells comprising the polynucleotides, and methods of producingsuch chimeric and hybrid polypeptides, and the polypeptides produced bysuch methods.

The present invention also provide a chimeric polypeptide that hasFactor VIII activity comprising a Factor VIII portion and a secondportion, wherein the Factor VIII portion is processed Factor VIIIcomprising two chains, a first chain comprising a heavy chain and asecond chain comprising a light chain, wherein said first chain and saidsecond chain are associated by a metal bond. For example, at least about50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 99% of the Factor VIII portion of the chimericpolypeptide is processed Factor VIII.

In addition, the present invention includes a chimeric polypeptide thathas Factor VIII activity, wherein the Factor VIII portion is singlechain Factor VIII. In one aspect, the single chain Factor VIII cancontain an intact intracellular processing site. In one embodiment, atleast about 1%, about 5%, about 10%, about 15%, about 20%, or about 25%of the Factor VIII portion of the chimeric polypeptide is single chainFactor VIII. In another embodiment, at least about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%of the Factor VIII portion of the chimeric polypeptide is single chainFactor VIII. In another aspect, the single chain FVIII does not containan intracellular processing site. For example, the SCFVIII comprises asubstitution or mutation at an amino acid position corresponding toArginine 1645, a substitution or mutation at an amino acid positioncorresponding to Arginine 1648, or a substitution or mutation at aminoacid positions corresponding to Arginine 1645 and Arginine 1648 infull-length Factor VIII. The amino acid substituted at the amino acidposition corresponding to Arginine 1645 is a different amino acid fromthe amino acid substituted at the amino acid position corresponding toArginine 1648. In certain embodiments, the substitution or mutation isan amino acid other than arginine, e.g., alanine.

In some embodiments, the chimeric polypeptide comprising single chainFactor VIII has Factor VIII activity at a level comparable to a chimericpolypeptide consisting of two Fc portions and processed Factor VIII,which is fused to one of the two Fc portions, when the Factor VIIIactivity is measured in vitro by a chromogenic assay. In otherembodiments, the chimeric polypeptide comprising single chain FactorVIII has Factor VIII activity in vivo comparable to a chimericpolypeptide consisting of two Fc portions and processed Factor VIII,which is fused to one of the two Fc portions. In still otherembodiments, the chimeric polypeptide comprising single chain FactorVIII has a Factor Xa generation rate comparable to a chimericpolypeptide consisting of two Fc portions and processed Factor VIII,which is fused to one of the two Fc portions. In certain embodiments,single chain Factor VIII in the chimeric polypeptide is inactivated byactivated Protein C at a level comparable to processed Factor VIII in achimeric polypeptide consisting of two Fc portions and processed FactorVIII. In yet other embodiments, the single chain Factor VIII in thechimeric polypeptide has a Factor IXa interaction rate comparable toprocessed Factor VIII in a chimeric polypeptide consisting of two Fcportions and processed Factor VIII. In further embodiments, the singlechain Factor VIII in the chimeric polypeptide binds to von WillebrandFactor at a level comparable to processed Factor VIII in a chimericpolypeptide consisting of two Fc portions and the processed Factor VIII.

The present invention further includes a composition comprising achimeric polypeptide having Factor VIII activity, wherein at least about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% ofsaid polypeptide comprises a Factor VIII portion, which is single chainFactor VIII, and a second portion, wherein said single chain Factor VIIIis at least 90% or 95% identical to a Factor VIII amino acid sequenceshown in Table 2 without a signal sequence (amino acids 20 to 1457 ofSEQ ID NO:2 or amino acids 20 to 2351 of SEQ ID NO:6). In oneembodiment, the second portion can be an Fc. In another embodiment, thepolypeptide is in the form of a hybrid comprising a second polypeptide,wherein said second polypeptide consists essentially of an Fc. In otherembodiments, the polypeptide has a half-life at least one and one-halfto six times longer, one and one-half to five times longer, one andone-half to four times longer, one and one-half to three times longer,or one and one-half to two times longer to a polypeptide consisting ofsaid Factor VIII.

Also provided is a method of treating a bleeding condition comprisingadministering a therapeutically effective amount of the composition. Thetreatment can be prophylactic treatment or on-demand treatment orperioperative. The bleeding coagulation disorder can be hemophilia. Inone embodiment, the subject that is treated is a pediatric subject.

The present invention is also directed to a method of preventing,decreasing, or treating a bleeding episode in a subject comprisingadministering to the subject an effective amount of a long-acting FactorVIII (FVIII) protein, wherein the subject expresses a high level of vonWillebrand Factor (VWF) in plasma. In one embodiment, the subject hasbeen identified as expressing a high level of VWF in plasma. The presentinvention is also directed to a method of preventing, decreasing, ortreating a bleeding episode in a subject comprising: (a) identifying asubject having high levels of VWF by measuring the level of VWF in theplasma of said subject, wherein a VWF level of at least about 100 IU/dLidentifies the subject as having a high level of VWF; and (b)administering to the subject an effective amount of a long-acting FVIIIprotein.

In one embodiment, the subject is a human. In another embodiment, thesubject is a pediatric subject. In another embodiment, the subject hashemophilia A.

In one embodiment, the high level of VWF is at least about 100 IU/dL. Inanother embodiment, the high level of VWF is between about 100 IU/dL andabout 200 IU/dL. In another embodiment, the high level of VWF is about110 IU/dL, about 120 IU/dL, about 130 IU/dL, about 140 IU/dL, about 150IU/dL, about 160 IU/dL, about 170 IU/dL, about 180 IU/dL, about 190IU/dL, or about 200 IU/dL.

In one embodiment the subject has the blood serotype A, B, or AB.

In one embodiment, the long-acting FVIII protein has a half-life in saidsubject of between about 20 and about 40 hours. In another embodiment,the long-acting FVIII protein has a half-life of about 21 hours, 22hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29hours, 30 hours, 31 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. In anotherembodiment, the long-acting FVIII protein has a half-life of betweenabout 20 and 27 hours. In another embodiment, the long-acting FVIIIprotein has a half-life that is at least about 1.2 times greater thanthe half-life of said said long-acting FVIII protein when administeredto an individual having average levels of VWF. In another embodiment,the long-acting FVIII protein has a half-life that is at least aboutabout 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or2.5-fold times greater than the half-life of said said long-acting FVIIIprotein when administered to an individual having average levels of VWF.

In one embodiment, the effective amount of long-acting FVIII proteinthat is administered is at least about 20 IU/kg, at least about 25IU/kg, at least about 30 IU/kg, at least about 35 IU/kg, at least about40 IU/kg, at least about 45 IU/kg, at least about 50 IU/kg, at leastabout 55 IU/kg, at least about 60 IU/kg, at least about 65 IU/kg, atleast about 70 IU/kg, at least about 75 IU/kg, at least about 80 IU/kg,at least about 85 IU/kg, or at least about 90 IU/kg. In anotherembodiment, the effective amount is at least about 65 IU/kg to at leastabout 90 IU/kg. In another embodiment, the effective amount is 80 IU/kg.

In one embodiment, the long-acting FVIII protein is administered every72 hours or longer. In another embodiment, the long-acting FVIII proteinis administered about once a week or longer. In another embodiment, thelong-acting FVIII protein is administered about once every 10 days,about once every two weeks, about once every 15 days, about once every20 days, about once every three weeks, about once every 25 days, aboutonce every four weeks, or about once every one month.

In one embodiment, the long-acting FVIII is administered at a dosage of80 IU/kg once every 72 hours. In a further embodiment the long-actingFVIII is administered at a dosage of 80 IU/kg once every 72 hours to apediatric subject.

In one embodiment, administration of the long-acting FVIII proteinresolves greater than 5-20%, greater than 5-15%, greater than 5-10%,greater than 10-20%, or greater than 10-15% of bleeding episodes. In oneembodiment, the trough level of plasma Factor VIII:C in the subjects ismaintained above 1-3 or 3-5 IU/dl. In one embodiment, the administrationprevents a bleeding episode in the subject. In another embodiment, thebleeding episode is spontaneous. In another embodiment, theadministration resolves greater than 80-100%, greater than 80-90%,greater than 85-90%, greater than 90-100%, greater than 90-95%, orgreater than 95-100% of bleeding episodes.

In one embodiment, the administration maintains homeostatis in thepopulation of the subjects in need of a surgery. In another embodiment,the long-acting FVIII protein is administered prior to, during, or afterthe surgery. In another embodiment, the surgery is minor surgery, majorsurgery, tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, total knee replacement, craniotomy, osteosynthesis, traumasurgery, intracranial surgery, intra-abdominal surgery, intrathoracicsurgery, or joint replacement surgery. In another embodiment, thesurgery is an emergency surgery.

In one embodiment, the long-acting FVIII protein has a half-life longerthan a polypeptide consisting of FVIII. In another embodiment, thelong-acting FVIII protein is pegylated, hesylated, or polysialylated.

In one embodiment, the long-acting FVIII protein is a chimeric proteincomprising a FVIII portion and a second portion. In another embodiment,the second portion is an Fc region, albumin, a PAS sequence,transferrin, CTP (28 amino acid C-terminal peptide (CTP) of hCG with its4 O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES),albumin binding polypeptide, albumin-binding small molecules, or two ormore combinations thereof. In another embodiment, the second portion isfused to the amino-terminus or the carboxy-terminus of the FVIIIportion. In another embodiment, the second portion is inserted betweentwo amino acids in the FVIII portion. In another embodiment, thechimeric protein is a FVIIIFc monomer dimer hybrid. In anotherembodiment, the FVIII portion is a single chain. In another embodiment,the FVIII portion comprises a heavy chain and a light chain. In anotherembodiment, the FVIII portion comprises full-length factor VIII, maturefactor VIII, or factor VIII with a full or partial deletion of the Bdomain. In another embodiment, the FVIII portion comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to2332 of SEQ ID NO: 6. In another embodiment, the FVIII portion comprisesamino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ IDNO: 6. In another embodiment, the chimeric polypeptide comprises an Fcregion which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids2333 to 2559 of SEQ ID NO: 6. In another embodiment, the second portioncomprises amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333to 2559 of SEQ ID NO: 6. In another embodiment, the long-acting FVIIIpolypeptide is administered as part of a pharmaceutical compositioncomprising at least one excipient.

The invention also provides a method of treating a subject diagnosedwith bleeding disorder, comprising measuring the half-life of FVIII-Fcin said subject, wherein a half-life that is at least about 1.2 timesgreater than the half-life of FVIII-Fc in a normal subject indicates thesubject is a candidate for long interval dosing, and administering aFVIII-Fc polypeptide in an effective amount and at a dosing interval ofat least 3 days.

The invention also provides a method of treating a subject diagnosedwith bleeding disorder, comprising administering a FVIII-Fc polypeptidein an effective amount and at a dosing interval of at least 3 days to asubject, wherein the half-life of FVIII-Fc in said subject is at leastabout 1.2 times greater than the half-life of FVIII-Fc when administeredto a subject having average levels of VWF.

In one embodiment, the plasma half-life of FVIII-Fc in said subject isat least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, or 2.5-fold times greater than the plasma half-life of FVIII-Fcwhen administered to a subject having average levels of VWF. In anotherembodiment, the FVIII-Fc plasma half-life is between 20-40 hours. Inanother embodiment, the long-acting FVIII protein has a half-life ofabout 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27hours, 28 hours, 29 hours, 30 hours, 31 hours, 31 hours, 32 hours, 33hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40hours. In another embodiment, the long-acting FVIII protein has ahalf-life of between about 20 and 27 hours.

The invention also provides a method of treating a subject diagnosedwith bleeding disorder, comprising measuring the half-life of ashort-acting FVIII administered to said subject, wherein a half-lifethat is at least about 1.2 times greater than the half-life of saidshort-acting FVIII in a subject having average VWF levels indicates thatthe subject is a candidate for long interval dosing, and administering along-acting FVIII-Fc polypeptide in an effective amount and at a dosinginterval of at least 3 days. In one embodiment, the half-life of theshort-acting FVIII in said subject is at least about 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold greater than thehalf-life of a short-acting FVIII when administered to a subject havingaverage levels of VWF.

In one embodiment, the subject is a human. In another embodiment, thesubject is a pediatric subject. In another embodiment, the subject hashemophilia A. In another embodiment, the subject has the blood serotypeA, B, or AB.

In one embodiment, the long-acting FVIII-Fc is administered in aneffective amount that is at least about 20 IU/kg, at least about 25IU/kg, at least about 30 IU/kg, at least about 35 IU/kg, at least about40 IU/kg, at least about 45 IU/kg, at least about 50 IU/kg, at leastabout 55 IU/kg, at least about 60 IU/kg, at least about 65 IU/kg, atleast about 70 IU/kg, at least about 75 IU/kg, at least about 80 IU/kg,at least about 85 IU/kg, or at least about 90 IU/kg. In anotherembodiment, the effective amount is at least about 65 IU/kg to at leastabout 90 IU/kg.

In one embodiment, the effective amount of the FVIII-Fc protein isadministered about once every week, about once every 10 days, about onceevery two weeks, about once every 15 days, about once every 20 days,about once every three weeks, about once every 25 days, about once everyfour weeks, or about once every one month.

In one embodiment, the administration resolves greater than 5-20%,greater than 5-15%, greater than 5-10%, greater than 10-20%, or greaterthan 10-15% of bleeding episodes. In one embodiment, the trough level ofplasma Factor VIII:C in the subjects is maintained above 1-3 or 3-5IU/dl.

In one embodiment, the administration prevents a bleeding episode in thesubject. In one embodiment, the bleeding episode is spontaneous. In oneembodiment, the administration resolves greater than 80-100%, greaterthan 80-90%, greater than 85-90%, greater than 90-100%, greater than90-95%, or greater than 95-100% of bleeding episodes. In one embodiment,the administration maintains homeostatis in the population of thesubjects in need of a surgery. In one embodiment, the FVIII-Fc proteinis administered prior to, during, or after the surgery. In oneembodiment, the surgery is minor surgery, major surgery, toothextraction, tonsillectomy, inguinal herniotomy, synovectomy, total kneereplacement, craniotomy, osteosynthesis, trauma surgery, intracranialsurgery, intra-abdominal surgery, intrathoracic surgery, or jointreplacement surgery. In one embodiment the surgery is an emergencysurgery.

In one embodiment, the FVIII-Fc protein has a half-life longer than apolypeptide consisting of FVIII. In one embodiment, the FVIII-Fc proteinis pegylated, hesylated, or polysialylated. In one embodiment, theFVIII-Fc protein is a FVIIIFc monomer dimer hybrid. In one embodiment,the FVIII portion is a single chain. In one embodiment, the FVIIIportion comprises a heavy chain and a light chain. In one embodiment,the FVIII portion comprises full-length factor VIII, mature factor VIII,or factor VIII with a full or partial deletion of the B domain. In oneembodiment, the FVIII portion comprises an amino acid sequence at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. Inone embodiment, the FVIII portion comprises amino acids 1 to 1438 of SEQID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. In one embodiment,the second portion of the chimeric polypeptide comprises an Fc regionwhich is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids2333 to 2559 of SEQ ID NO: 6. In one embodiment, the second portioncomprises amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333to 2559 of SEQ ID NO: 6.

In one embodiment, the FVIII-Fc polypeptide is administered as part of apharmaceutical composition comprising at least one excipient.

The invention also provides a method for determining whether a subjectdiagnosed with bleeding disorder is a candidate for long interval dosingwith a long-acting FVIII polypeptide, comprising measuring theexpression levels of plasma VWF, wherein an VWF expression level of atleast 100 IU/dL indicates that the subject is a candidate for longinterval dosing using a long-acting FVIII polypeptide. In oneembodiment, the VWF expression level is at least about 110 IU/dL, about120 IU/dL, about 130 IU/dL, about 140 IU/dL, about 150 IU/dL, about 160IU/dL, about 170 IU/dL, about 180 IU/dL, about 190 IU/dL, or about 200IU/dL.

The invention also provides a method for determining whether a subjectdiagnosed with bleeding disorder is a candidate for long interval dosingof a long-acting FVIII polypeptide, comprising measuring the half-lifeof FVIII-Fc in said subject, wherein a half-life that is at least about1.2-fold greater than the half-life of FVIII-Fc when administered to asubject having average VWF levels indicates the subject is a candidatefor long interval dosing. In one embodiment, the half-life of FVIII-Fcis at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, or 2.5-fold greater than the half-life of FVIII-Fc whenadministered to a subject having average levels of VWF.

The invention also provides a method for determining whether a subjectdiagnosed with bleeding disorder is a candidate for long interval dosingof a long-acting FVIII polypeptide, comprising measuring the half-lifeof short-acting FVIII in said subject, wherein a half-life that is atleast about 1.2-fold greater than the half-life of short-acting FVIIIwhen administered to a subject having average VWF levels indicates thesubject is a candidate for long interval dosing. In one embodiment, thehalf-life is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, or 2.5-fold greater than the half-life of FVIII-Fc whenadministered to a subject having average levels of VWF.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Schematic Representation of rFVIIIFc monomer.

FIGS. 2A-E. A-B. Non-reducing and reducing SDS-PAGE analysis of rFVIIIFc(processed or single chain). C. rFVIIIFc structure analyzed by LC/UV andLC/MS. D. Total Ion Current (TIC) chromatogram (LC/MS map) of rFVIIIFcafter thrombin cleavage. Major digestion products are indicated. E.Deconvoluted Mass Spectrum of the A2 domain of rFVIIIFc and rBDDFVIII.Major products and their cognate masses are indicated, corresponding tothrombin-cleaved A2 domain (S373 to R740) and two truncated products,S373 to Y729 and S373 to E720.

FIGS. 3A-C. Biochemical characterization of rFVIII-Fc: A. Activation ofFactor X as a function of phospholipid vesicle concentration; B.Activation of Factor X as a function of FX concentration. C. Activationof Factor X as a function of Factor IXa concentration.

FIG. 4. Activation of Factor X following cleavage by activated ProteinC.

FIGS. 5A-D. Observed group mean FVIII activity (±SE) versus timeprofiles, sorted by dose level, grouped by compound (one stage assay, 25IU/kg (A) or 65 IU/kg (B); and chromogenic assay, 25 IU/kg (C) or 65IU/kg (D)) versus time.

FIGS. 6A-B. Observed group mean FVIII activity (±SE) versus timeprofiles, grouped by dose level and compound (one stage assay (A) orchromogenic assay (B)) versus time.

FIGS. 7(A)-(C). In Vivo Efficacy of Single Chain FVIII:Fc in HemA MouseTail Vein Transection Model. (A) Single chain rFVIII:Fc doses are shownas squares, and processed rFVIII:Fc doses are shown as circles. (B)Percent survival following tail vein transection of 4.6 μg/kg, 1.38μg/kg, and 0.46 μg/kg of rFVIIIFc or SC rFVIIIFc. (C) Percent ofnon-bleeders following tail vein transection of 4.6 μg/kg (black circleor inverted triangle), 1.38 μg/kg (triangle or diamond), and 0.46 μg/kg(square and gray circle) of rFVIIIFc or SC rFVIIIFc, respectively.

FIG. 8. Study Design. FIG. 8 depicts the study design of the phase 1/2astudy, which was a dose-escalation, sequential design to evaluate thesafety and PK of rFVIIIFc compared with ADVATE® after a singleintravenous dose of either 25 IU/kg (low dose Cohort A) or 65 IU/kg(high dose Cohort B).

FIG. 9. Correlation of rFVIII Activity by One-Stage (aPTT) andChromogenic Assays. Correlation between one-stage clotting (aPTT) andchromogenic assay results measuring FVIII activity (IU/mL) followinginjection of ADVATE® (♦) and rFVIIIFc (□).

FIGS. 10(A)-(B). Group Mean Plasma FVIII Activity PharmacokineticProfiles for Low-Dose and High-Dose Cohorts. The plasma FVIII activity(one stage aPTT assay) versus time curve after a single intravenousinjection of rFVIIIFc or ADVATE® are shown for (A) 25 IU/kg (low-dosecohort, n=6); and (B) 65 IU/kg (high dose cohort, n=10 [ADVATE®]; n=9[rFVIIIFc]). Results presented are group mean±standard error of mean(SEM).

FIGS. 11(A)-(B). Effect of VWF Antigen Levels on C1 and t_(1/2) of FVIIIActivity after Injection of ADVATE® or rFVIIIFc. Correlation between VWFantigen levels and (A) the weight-adjusted C1 of ADVATE® (R²=0.5415 andp=0.0012) and rFVIIIFc (R²=0.5492 and p=0.0016); and (B) the t_(1/2) ofADVATE® (R²=0.7923 and p<0.0001) and rFVIIIFc (R²=0.6403 and p=0.0003).Each dot represents an individual subject.

FIGS. 12(A)-(B). Ex Vivo Whole Blood ROTEM® Results for IndividualSubjects After Injection of ADVATE® or rFVIIIFc. Blood was sampled fromsubjects prior to and after treatment at doses of (A) 25 IU/kg ADVATE®and rFVIIIFc; and (B) 65 IU/kg ADVATE® and rFVIIIFc at specified timepoints. Clotting time was determined by NATEM initiated with Ca++ on aROTEM® instrument. Results presented are mean±standard error of mean(SEM) from triplicate channel readings for each individual sample.

FIGS. 13(A)-(B). Activity comparison in thrombin generation assay (TGA).(A) SC rFVIIIFc showed a reduced endogenous thrombin potential (ETP),and (B) a reduced peak thrombin compared to rFVIIIFc.

FIGS. 14(A)-(C): In vitro ROTEM data. ROTEM (NATEM) results (Mean±SD)for varying concentratios of XYNTHA, ADVATE< and rFVIIIFc spked inpooled whole blood obtained from naïve HemA mice. (A). Average clot time(CT) (FIG. 14A), (B). clot formation time (CFT), and (C). alpha angle.

FIGS. 15(A)-(C). Ex vivo ROTEM data. ROTEM (NATEM) results (Mean±SD)from HemA mice following a single intravenous administration of 50 IU/kgof XYNTHA, ADVATE, or rFVIIIFc at 5 min, 24, 48, 72, and 96 hours afterdosing. (A). Average clot time (CT), (B). Clot formation time (CFT), and(C). alpha angle.

FIGS. 16(A)-(E): Real-time evaluation of the interaction of rFVIIIFc andsingle chain (SC) rFVIIIFc with vWF, and real-time evaluation ofthrombin mediated release of rFVIIIFc and SC rFVIIIFc from vWF. (A).Surface plasmon resonance (SPR) analysis of rFVIIIFc and SC rFVIIIFcaffinity for vWF. Depicted are the binding curve and the 1:1 fitinteraction model. The x-axis shows time in seconds and the y-axis showsresponse in response units (RU). (B). Reference subtracted sensograms ofthrombin-mediated release of activated rFVIIIFc, SC rFVIIIFc, andB-domain deleted rFVIII lacking Fc moieties (rBDD FVIII) at 25° C. (top)and 37° C. (bottom). The x-axis shows time in seconds and the y-axisshows response in response units (RU). Individual lines indicate theresponse at different α-thrombin concentrations. The uppermost line isthe response at 0 U/mL α-thrombin, and each subsequent line runs inorder for α-thrombin concentrations of 0.005, 0.01, 0.02, 0.04, 0.08,0.16, 0.31, 0.63, 1.3, 2.5, 5, 10, and 20 U/mL. (C). Double referencesubtracted sensograms of thrombin mediated release phase for rFVIIIFc,SC rFVIIIFc, and rBDD FVIII at 25° C. (top) and 37° C. (bottom). Thex-axis shows time in seconds and the y-axis shows response in responseunits (RU). Individual lines indicate response at different α-thrombinconcentrations. The uppermost line is the response at 0 U/mL α-thrombin,and each subsequent line runs in order for α-thrombin concentrations of0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.3, 2.5, 5.0, 10, and20 U/mL. (D). Thrombin-mediated release rate as a function of time forrFVIIIFc, SC rFVIIIFc, and rBDD FVIII at 25° C. (top) and 37° C.(bottom). The x-axis shows time in seconds and the y-axis shows responsein response units (RU). Individual lines indicate response at differentα-thrombin concentrations. The uppermost line is the response at 20 U/mLα-thrombin, and each subsequent line runs in order for α-thrombinconcentrations of 10, 5, 2.5, 1.3, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02,0.01, and 0.005 U/mL. (E). Peak thrombin-mediated release rate as afunction of thrombin concentration for rFVIIIFc, SC rFVIIIFc, and rBDDFVIII at 25° C. (top) and 37° C. (bottom). EC₅₀ is half maximaleffective concentration. The x-axis is α-thrombin concentration in U/mLand the y-axis is maximum release rate in RU/second.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating Hemophilia A withFactor VIII (processed, single chain, or a combination thereof) using alonger dosing interval and/or greater AUC than is possible withcurrently known Factor VIII products. The present invention alsoprovides improved Factor VIII chimeric polypeptides and methods ofproduction.

Treatment of hemophilia A is by replacement therapy targetingrestoration of FVIII activity to 1 to 5% of normal levels to preventspontaneous bleeding (Mannucci, P. M. et al., N. Engl. J. Med.344:1773-9 (2001), herein incorporated by reference in its entirety).There are plasma-derived and recombinant FVIII products available totreat bleeding episodes on-demand or to prevent bleeding episodes fromoccurring by treating prophylactically. Based on the short half-life ofthese products (8-12 hr) (White G. C., et al., Thromb. Haemost. 77:660-7(1997); Morfini, M., Haemophilia 9 (suppl 1):94-99; discussion 100(2003)), treatment regimens require frequent intravenous administration,commonly two to three times weekly for prophylaxis and one to threetimes daily for on-demand treatment (Manco-Johnson, M. J., et al., N.Engl. J. Med. 357:535-544 (2007)), each of which is incorporated hereinby reference in its entirety. Such frequent administration is painfuland inconvenient.

The present invention provides a method of administering Factor VIII toa human subject in need thereof (e.g., human patient), comprisingadministering to the subject a therapeutic dose of a chimeric FactorVIII polypeptide, e.g., a chimeric Factor VIII-Fc polypeptide, or ahybrid of such a polypeptide at a dosing interval at least about one andone-half times longer than the dosing interval required for anequivalent dose of said Factor VIII without the non-Factor VIII portion(a polypeptide consisting of said Factor VIII portion), e.g., withoutthe Fc portion. The present invention is also directed to a method ofincreasing dosing interval of Factor VIII administration in a humansubject in need thereof comprising administering the chimeric FactorVIII polypeptide.

The dosing interval may be at least about one and one-half to six timeslonger, one and one-half to five times longer, one and one-half to fourtimes longer, one and one-half to three times longer, or one andone-half to two times longer, than the dosing interval required for anequivalent dose of said Factor VIII without the non-Factor VIII portion(a polypeptide consisting of said Factor VIII portion), e.g., withoutthe Fc portion. The dosing interval may be at least about one andone-half, two, two and one-half, three, three and one-half, four, fourand one-half, five, five and one-half or six times longer than thedosing interval required for an equivalent dose of said Factor VIIIwithout the non-Factor VIII portion (a polypeptide consisting of saidFactor VIII portion), e.g., without the Fc portion. The dosing intervalmay be about every three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, or fourteen days or longer.

The dosing interval may be at least about one and one-half to 5, one andone-half, 2, 3, 4, or 5 days or longer.

The present invention also provides a method of administering FactorVIII to a human subject in need thereof, comprising administering to thesubject a therapeutic dose of a chimeric Factor VIII polypeptide, e.g.,a chimeric Factor VIII-Fc polypeptide, or a hybrid of such a polypeptideto obtain an area under the plasma concentration versus time curve (AUC)at least about one and one-quarter times greater than the AUC obtainedby an equivalent dose of said Factor VIII without non-Factor VIIIportion (a polypeptide consisting of said Factor VIII portion), e.g.,without the Fc portion. The present invention thus includes a method ofincreasing or extending AUC of Factor VIII activity in a human patientin need thereof comprising administering the chimeric Factor VIIIpolypeptide.

The present invention also provides a method of administering FactorVIII to a subject in need thereof, comprising administering to thesubject a therapeutic dose of a polypeptide comprising a Factor VIII andan Fc or a hybrid of such a polypeptide at a dosing interval of aboutevery three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, or fourteen days or longer.

The methods of the invention may be practiced on a subject in need ofprophylactic treatment or on-demand treatment.

“Administering,” as used herein, means to give a pharmaceuticallyacceptable Factor VIII polypeptide of the invention to a subject via apharmaceutically acceptable route. Routes of administration can beintravenous, e.g., intravenous injection and intravenous infusion.Additional routes of administration include, e.g., subcutaneous,intramuscular, oral, nasal, and pulmonary administration. Chimericpolypeptides and hybrid proteins may be administered as part of apharmaceutical composition comprising at least one excipient.

“Area under the plasma concentration versus time curve (AUC),” as usedherein, is the same as the term of art in pharmacology, and is basedupon the rate and extent of absorption of Factor VIII followingadministration. AUC is determined over a specified time period, such as12, 18, 24, 36, 48, or 72 hours, or for infinity using extrapolationbased on the slope of the curve. Unless otherwise specified herein, AUCis determined for infinity. The determination of AUC may be carried outin a single subject, or in a population of subjects for which theaverage is calculated.

A “B domain” of Factor VIII, as used herein, is the same as the B domainknown in the art that is defined by internal amino acid sequenceidentity and sites of proteolytic cleavage by thrombin, e.g., residuesSer741-Arg1648 of full length human factor VIII. The other human factorVIII domains are defined by the following amino acid residues: A1,residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residuesSer1690-Ile2032; C1, residues Arg2033-Asn2172; C2, residuesSer2173-Tyr2332. The A3-C1-C2 sequence includes residuesSer1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, isusually referred to as the factor VIII light chain activation peptide.The locations of the boundaries for all of the domains, including the Bdomains, for porcine, mouse and canine factor VIII are also known in theart. In one embodiment, the B domain of Factor VIII is deleted (“Bdomain deleted factor VIII” or “BDD FVIII”). An example of a BDD FVIIIis REFACTO® (recombinant BDD FVIII), which has the same sequence as theFactor VIII portion of the sequence in Table 2A(i) (amino acids 1 to1457 or 20 to 1457 of SEQ ID NO:2). In another embodiment, the B domaindeleted Factor VIII contains an intact intracellular processing site,which corresponds to Arginine at residue 754 of B domain deleted FactorVIII, which corresponds to Arginine residue 773 of SEQ ID NO: 2, orresidue 1648 of full-length Factor VIII, which corresponds to Arginineresidue 1667 of SEQ ID NO: 6. The sequence residue numbers used hereinwithout referring to any SEQ ID Numbers correspond to the Factor VIIIsequence without the signal peptide sequence (19 amino acids) unlessotherwise indicated. For example, S743/Q1638 of full-length Factor VIIIcorresponds to S762/Q1657 of SEQ ID NO: 6 due to the 19 amino acidsignal peptide sequence. In other embodiments, the B domain deletedFVIII comprises a substitution or mutation at an amino acid positioncorresponding to Arginine 1645, a substitution or mutation at an aminoacid position corresponding to Arginine 1648, or a substitution ormutation at amino acid positions corresponding to Arginine 1645 andArginine 1648 in full-length Factor VIII. In some embodiments, the aminoacid substituted at the amino acid position corresponding to Arginine1645 is a different amino acid from the amino acid substituted at theamino acid position corresponding to Arginine 1648. In certainembodiments, the substitution or mutation is an amino acid other thanarginine, e.g., alanine.

A “B domain deleted factor VIII” may have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of whichis incorporated herein by reference in its entirety. In someembodiments, a B domain deleted factor VIII sequence of the presentinvention comprises any one of the deletions disclosed at col. 4, line 4to col. 5, line 28 and examples 1-5 of U.S. Pat. No. 6,316,226 (also inU.S. Pat. No. 6,346,513). In some embodiments, a B domain deleted factorVIII of the present invention has a deletion disclosed at col. 2, lines26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. No.6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620). Insome embodiments, a B domain deleted factor VIII has a deletiondescribed in col. 1, lines 25 to col. 2, line 40 of U.S. Pat. No.5,972,885; col. 6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720;col. 2, lines 17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col.5, line 36 of U.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, andexample 1 of U.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21and table 2 of U.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line26, and col. 11, line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635;or col. 4, lines 25-53, of U.S. Pat. No. 6,458,563. In some embodiments,a B domain deleted factor VIII has a deletion of most of the B domain,but still contains amino-terminal sequences of the B domain that areessential for in vivo proteolytic processing of the primary translationproduct into two polypeptide chain (i.e., intracellular processingsite), as disclosed in WO 91/09122, which is incorporated herein byreference in its entirety. In some embodiments, a B domain deletedfactor VIII is constructed with a deletion of amino acids 747-1638,i.e., virtually a complete deletion of the B domain. Hoeben R. C., etal. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein byreference in its entirety. A B domain deleted factor VIII may alsocontain a deletion of amino acids 771-1666 or amino acids 868-1562 offactor VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988),incorporated herein by reference in its entirety. Additional B domaindeletions that are part of the invention include, e.g.,: deletion ofamino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc.Natl. Acad. Sci. U.S.A. 83:5939-5942 (1986)), 797 through 1562 (Eaton etal., Biochemistry 25:8343-8347 (1986)), 741 through 1646 (Kaufman (PCTpublished application No. WO 87/04187)), 747-1560 (Sarver et al., DNA6:553-564 (1987)), 741 through 1648 (Pasek (PCT application No.88/00831)), 816 through 1598 or 741 through 1689 (Lagner (Behring Inst.Mitt. (1988) No. 82:16-25, EP 295597)), each of which is incorporatedherein by reference in its entirety. Each of the foregoing deletions maybe made in any Factor VIII sequence.

In one embodiment, the B domain deleted Factor VIII portion in thechimeric polypeptide is processed into two chains connected (orassociated) by a metal bond, the first chain comprising a heavy chain(A1-A2-partial B) and a second chain comprising a light chain(A3-C1-C2). In another embodiment, the B domain deleted Factor VIIIportion is a single chain Factor VIII. The single chain Factor VIII cancomprise an intracellular processing site, which corresponds to Arginineat residue 754 of B domain deleted Factor VIII (residue 773 of SEQ IDNO: 2) or at residue 1648 of full-length Factor VIII (residue 1657 ofSEQ ID NO: 6).

The metal bond between the heavy chain and the light chain can be anymetal known in the art. For example, the metals useful for the inventioncan be a divalent metal ion. The metals that can be used to associatethe heavy chain and light chain include, but not limited to, Ca²⁺, Mn²⁺,or Cu²⁺. Fatouros et al., Intern. J. Pharm. 155(1): 121-131 (1997);Wakabayashi et al., JBC. 279(13): 12677-12684 (2004).

“Chimeric polypeptide,” as used herein, means a polypeptide thatincludes within it at least two polypeptides (or subsequences orpeptides) from different sources. Chimeric polypeptides may include,e.g., two, three, four, five, six, seven, or more polypeptides fromdifferent sources, such as different genes, different cDNAs, ordifferent animal or other species. Chimeric polypeptides may include,e.g., one or more linkers joining the different subsequences. Thus, thesubsequences may be joined directly or they may be joined indirectly,via linkers, or both, within a single chimeric polypeptide. Chimericpolypeptides may include, e.g., additional peptides such as signalsequences and sequences such as 6His and FLAG that aid in proteinpurification or detection. In addition, chimeric polypeptides may haveamino acid or peptide additions to the N- and/or C-termini.

In some embodiments, the chimeric polypeptide comprises a Factor VIIIportion and a non-Factor VIII portion. Exemplary non-Factor VIIIportions include, e.g., Fc, XTEN, albumin, a PAS sequence, transferrin,CTP (28 amino acid C-terminal peptide (CTP) of human chorionicgonadotropin (hCG) with its 4 O-glycans), polyethylene glycol (PEG),hydroxyethyl starch (HES), albumin binding polypeptide, andalbumin-binding small molecules. Exemplary chimeric polypeptides of theinvention include, e.g., chimeric Factor VIII-Fc polypeptides, chimericFactor VIII-XTEN polypeptides, chimeric Factor VIII-albuminpolypeptides, chimeric Factor VIII-PAS polypeptides, chimeric FactorVIII-transferrin polypeptides, chimeric Factor VIII-CTP polypeptides,chimeric Factor VIII-PEG polypeptides, chimeric Factor VIII-HESpolypeptides, chimeric Facotr VIII-albumbin binding polypeptidepolypeptides, and chimeric Factor VIII.-albumin-binding small moleculepolypeptides.

Exemplary chimeric Factor VIII-Fc polypeptides include, e.g., SEQ IDNO:2 or 6 (Table 2), with or without their signal sequences and thechimeric Fc polypeptide of SEQ ID NO:4 (Table 2).

The chimeric polypeptide may comprise a sequence at least 90% or 95%identical to the Factor VIII and Fc amino acid sequence shown in Table2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO:2)or at least 90% or 95% identical to the Factor VIII and Fc amino acidsequence shown in Table 2A(i) with a signal sequence (amino acids 1 to1684 of SEQ ID NO:2), wherein the sequence has Factor VIII activity. TheFactor VIII activity can be measured by activated Partial ThromboplastinTime (aPPT) assay, chromogenic assay, or other known methods. Thechimeric polypeptide may comprise a sequence identical to the FactorVIII and Fc amino acid sequence shown in Table 2A(i) without a signalsequence (amino acids 20 to 1684 of SEQ ID NO:2) or identical to theFactor VIII and Fc amino acid sequence shown in Table 2A(i) with asignal sequence (amino acids 1 to 1684 of SEQ ID NO:2).

As discussed above, exemplary chimeric polypeptides include Factor VIIIfused to one or more XTEN polypeptides. Schellenburger et al., Nat.Biotech. 27:1186-90 (2009), which is incorporated herein by reference inits entirety. The XTEN polypeptide can be fused to either the N-terminalend of FVIII or to the C-terminal end of FVIII. A protease site may beincluded between the XTEN portion and the Factor VIII portion to allowsuch processing. XTEN polypeptides include, e.g., those disclosed in WO2009/023270, WO 2010/091122, WO 2007/103515, US 2010/0189682, and US2009/0092582, each of which is incorporated herein by reference in itsentirety.

As discussed above, exemplary chimeric polypeptides also include FactorVIII fused to one or more albumin polypeptides, albumin bindingpolypeptides, or albumin-binding small molecules. In one embodiment, thealbumin is human albumin. The albumin or albumin binding protein can befused to either the N-terminal end of FVIII or to the C-terminal end ofFVIII or inserted between two amino acids in FVIII. Examples of albumin,e.g., fragments thereof, that may be used in the present invention areknown. e.g., U.S. Pat. No. 7,592,010; U.S. Pat. No. 6,686,179; andSchulte, Thrombosis Res. 124 Suppl. 2:S6-S8 (2009), each of which isincorporated herein by reference in its entirety.

The albumin binding polypeptides can compromise, without limitation,bacterial albumin-binding domains, albumin-binding peptides, oralbumin-binding antibody fragments that can bind to albumin. Domain 3from streptococcal protein G, as disclosed by Kraulis et al., FEBS Lett.378:190-194 (1996) and Linhult et al., Protein Sci. 11:206-213 (2002) isan example of a bacterial albumin-binding domain. Examples ofalbumin-binding peptides include a series of peptides having the coresequence DICLPRWGCLW (SEQ ID NO: 7). See, e.g., Dennis et al., J. Biol.Chem. 2002, 277: 35035-35043 (2002). Examples of albumin-bindingantibody fragments are disclosed in Muller and Kontermann, Curr. Opin.Mol. Ther. 9:319-326 (2007); Rooverset et al., Cancer Immunol.Immunother. 56:303-317 (2007), and Holt et al., Prot. Eng. Design Sci.,21:283-288 (2008), which are incorporated herein by reference in theirentireties.

In certain aspects, a recombinant FVIII polypeptide of the inventioncomprises at least one attachment site for a non-polypeptide smallmolecule, variant, or derivative that can bind to albumin thereof. Anexample of such albumin binding moieties is2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate(“Albu” tag) as disclosed by Trusselet et al., Bioconjugate Chem.20:2286-2292 (2009).

As discussed above, exemplary chimeric polypeptides also include FactorVIII fused to at least one β subunit of the C-terminal peptide (CTP) ofhuman chorionic gonadotropin or fragment, variant, or derivativethereof. The CTP can be fused to Factor VIII either the N-terminal endof FVIII or to the C-terminal end of FVIII or inserted between two aminoacids in FVIII. One or more CTP peptides fused to or inserted into arecombinant protein is known to increase the in vivo half-life of thatprotein. See, e.g., U.S. Pat. No. 5,712,122, incorporated by referenceherein in its entirety. Exemplary CTP peptides includeDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 8) orSSSSKAPPPSLPSPSRLPGPSDTPILPQ. (SEQ ID NO: 9). See, e.g., U.S. PatentApplication Publication No. US 2009/0087411 A1, incorporated byreference.

As discussed above, exemplary chimeric polypeptides also include FactorVIII fused to at least one PAS sequence or fragment, variant, orderivative thereof. The PAS sequence can be fused to either theN-terminal end of FVIII or to the C-terminal end of FVIII or insertedbetween two amino acids in FVIII. A PAS peptide or PAS sequence, as usedherein, means an amino acid sequence comprising mainly alanine andserine residues or comprising mainly alanine, serine, and prolineresidues, the amino acid sequence forming random coil conformation underphysiological conditions. Accordingly, the PAS sequence is a buildingblock, an amino acid polymer, or a sequence cassette comprising,consisting essentially of, or consisting of alanine, serine, and prolinewhich can be used as a part of the heterologous moiety in the chimericprotein. An amino acid polymer also can form random coil conformationwhen residues other than alanine, serine, and proline are added as aminor constituent in the PAS sequence. By “minor constituent” is meantthat that amino acids other than alanine, serine, and proline can beadded in the PAS sequence to a certain degree, e.g., up to about 12%,i.e., about 12 of 100 amino acids of the PAS sequence, up to about 10%,up to about 9%, up to about 8%, about 6%, about 5%, about 4%, about 3%,i.e. about 2%, or about 1%, of the amino acids. The amino acidsdifferent from alanine, serine and proline cab be selected from thegroup consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Thr, Trp, Tyr, and Val. Under physiological conditions, aPAS peptide forms a random coil conformation and thereby can mediate anincreased in vivo and/or in vitro stability to a recombinant protein ofthe invention, and has procoagulant activity.

Non-limiting examples of the PAS peptides include ASPAAPAPASPAAPAPSAPA(SEQ ID NO: 10), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 11),APSSPSPSAPSSPSPASPSS (SEQ ID NO: 12), APSSPSPSAPSSPSPASPS (SEQ ID NO:13), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 14), AASPAAPSAPPAAASPAAPSAPPA (SEQID NO: 15), ASAAAPAAASAAASAPSAAA (SEQ ID NO: 16) or any variants,derivatives, fragments, or combinations thereof. Additional examples ofPAS sequences are known from, e.g., US Pat. Publ. No. 2010/0292130 A1and PCT Appl. Publ. No. WO 2008/155134 A1. European issued patentEP2173890.

As discussed above, exemplary chimeric polypeptides also include FactorVIII fused to at least one transferrin peptide or fragment, variant, orderivative thereof. At least one transferrin peptide can be fused toeither the N-terminal end of FVIII or to the C-terminal end of FVIII orinserted between two amino acids in FVIII. Any transferrin can be fusedto or inserted into a recombinant FVIII protein of the invention. As anexample, wild-type human Tf (Tf) is a 679 amino acid protein, ofapproximately 75 KDa (not accounting for glycosylation), with two maindomains, N (about 330 amino acids) and C (about 340 amino acids), whichappear to originate from a gene duplication. See GenBank accessionnumbers NM001063, XM002793, M12530, XM039845, XM 039847 and S95936(www.ncbi.nlm.nih.gov), all of which are herein incorporated byreference in their entirety.

Transferrin transports iron through transferrin receptor (TfR)-mediatedendocytosis. After the iron is released into an endosomal compartmentand Tf-TfR complex is recycled to cell surface, the Tf is released backextracellular space for next cycle of iron transporting. Tf possesses along half-life that is in excess of 14-17 days (Li et al., TrendsPharmacol. Sci. 23:206-209 (2002)). Transferrin fusion proteins havebeen studied for half-life extension, targeted deliver for cancertherapies, oral delivery and sustained activation of proinsulin(Brandsma et al., Biotechnol. Adv., 29: 230-238 (2011); Bai et al.,Proc. Natl. Acad. Sci. USA 102:7292-7296 (2005); Kim et al., J.Pharmacol. Exp. Ther., 334:682-692 (2010); Wang et al., J. ControlledRelease 155:386-392 (2011)).

As discussed above, exemplary chimeric polypeptides also include FactorVIII fused to at least one polyethylene glycol (PEG) moieties.

PEGylated FVIII can refer to a conjugate formed between FVIII and atleast one polyethylene glycol (PEG) molecule. PEG is commerciallyavailable in a large variety of molecular weights and average molecularweight ranges. Typical examples of PEG average molecular weight rangesinclude, but are not limited to, about 200, about 300, about 400, about600, about 1000, about 1300-1600, about 1450, about 2000, about 3000,about 3000-3750, about 3350, about 3000-7000, about 3500-4500, about5000-7000, about 7000-9000, about 8000, about 10000, about 8500-11500,about 16000-24000, about 35000, about 40000, about 60000, and about80000 daltons. These average molecular weights are provided merely asexamples and are not meant to be limiting in any way.

A recombinant FVIII protein of the invention can be PEGylated to includemono- or poly- (e.g., 2-4) PEG moieties. PEGylation can be carried outby any of the PEGylation reactions known in the art. Methods forpreparing a PEGylated protein product will generally include (i)reacting a polypeptide with polyethylene glycol (such as a reactiveester or aldehyde derivative of PEG) under conditions whereby thepeptide of the invention becomes attached to one or more PEG groups; and(ii) obtaining the reaction product(s). In general, the optimal reactionconditions for the reactions will be determined case by case based onknown parameters and the desired result.

There are a number of PEG attachment methods available to those skilledin the art, for example Malik F et al., Exp. Hematol. 20:1028-35 (1992);Francis, Focus on Growth Factors 3(2):4-10 (1992); European Pat. Pub.Nos. EPO401384, EP0154316, and EPO401384; and International Pat. Appl.Pub. Nos. WO92/16221 and WO95/34326. As a non-limiting example, FVIIIvariants can contain cysteine substitutions in one or more insertionsites in FVIII, and the cysteines can be further conjugated to PEGpolymer. See Mei et al., Blood 116:270-279 (2010) and U.S. Pat. No.7,632,921, which are incorporated herein by reference in theirentireties.

As discussed above, exemplary chimeric polypeptides also include FactorVIII fused to at least one hydroxyethyl starch (HES) polymer. HES is aderivative of naturally occurring amylopectin and is degraded byalpha-amylase in the body. HES exhibits advantageous biologicalproperties and is used as a blood volume replacement agent and inhemodilution therapy in the clinics. See, e.g., Sommermeyer et al.,Krankenhauspharmazie 8:271-278 (1987); and Weidler et al.,Arzneim.-Forschung/Drug Res. 41: 494-498 (1991).

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. HES has a mean molecular weight (weight mean) offrom 1 to 300 kD, from 2 to 200 kD, from 3 to 100 kD, or from 4 to 70kD. Hydroxyethyl starch can further exhibit a molar degree ofsubstitution of from 0.1 to 3, from 0.1 to 2, from 0.1 to 0.9, or from0.1 to 0.8, and a ratio between C2:C6 substitution in the range of from2 to 20 with respect to the hydroxyethyl groups. HES with a meanmolecular weight of about 130 kD is Voluven® from Fresenius. Voluven® isan artificial colloid, employed, e.g., for volume replacement used inthe therapeutic indication for therapy and prophylaxis of hypovolaemia.There are a number of HES attachment methods available to those skilledin the art, e.g., the same PEG attachment methods described above.

In some embodiments, a chimeric polypeptide comprising a Factor VIIIportion has an increased half-life (t1/2) over a polypeptide consistingof the same Factor VIII portion without the non Factor VIII portion. Achimeric Factor VIII polypeptide with an increased t1/2 may be referredto herein as a long-acting Factor VIII. Long-acting chimeric Factor VIIIpolypeptides include, e.g., Factor VIII fused to Fc (including, e.g.,chimeric Factor VIII polypeptides in the form of a hybrid such as aFVIIIFc monomer dimer hybrid; see Example 1, FIG. 1, and Table 2A; andU.S. Pat. Nos. 7,404,956 and 7,348,004), Factor VIII fused to XTEN, andFactor VIII fused to albumin.

“Culture,” “to culture” and “culturing,” as used herein, means toincubate cells under in vitro conditions that allow for cell growth ordivision or to maintain cells in a living state. “Cultured cells,” asused herein, means cells that are propagated in vitro.

“Factor VIII,” as used herein, means functional factor VIII polypeptidein its normal role in coagulation, unless otherwise specified. Thus, theterm Factor VIII includes variant polypeptides that are functional.Factor VIII proteins can be the human, porcine, canine, and murinefactor VIII proteins. As described in the Background Art section, thefull length polypeptide and polynucleotide sequences are known, as aremany functional fragments, mutants and modified versions. Examples ofhuman factor VIII sequences are shown as subsequences in SEQ ID NOs:2 or6 (Table 2). Factor VIII polypeptides include, e.g., full-length factorVIII, full-length factor VIII minus Met at the N-terminus, mature factorVIII (minus the signal sequence), mature factor VIII with an additionalMet at the N-terminus, and/or factor VIII with a full or partialdeletion of the B domain. Factor VIII variants include B domaindeletions, whether partial or full deletions.

A great many functional factor VIII variants are known, as is discussedabove and below. In addition, hundreds of nonfunctional mutations infactor VIII have been identified in hemophilia patients, and it has beendetermined that the effect of these mutations on factor VIII function isdue more to where they lie within the 3-dimensional structure of factorVIII than on the nature of the substitution (Cutler et al., Hum. Mutat.19:274-8 (2002)), incorporated herein by reference in its entirety. Inaddition, comparisons between factor VIII from humans and other specieshave identified conserved residues that are likely to be required forfunction (Cameron et al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat.No. 6,251,632), incorporated herein by reference in its entirety.

The human factor VIII gene was isolated and expressed in mammalian cells(Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al.,Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337(1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), each of which isincorporated herein by reference in its entirety, and the amino acidsequence was deduced from cDNA. Capon et al., U.S. Pat. No. 4,965,199,incorporated herein by reference in its entirety, discloses arecombinant DNA method for producing factor VIII in mammalian host cellsand purification of human factor VIII. Human factor VIII expression inCHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells)has been reported. Human factor VIII has been modified to delete part orall of the B domain (U.S. Pat. Nos. 4,994,371 and 4,868,112, each ofwhich is incorporated herein by reference in its entirety), andreplacement of the human factor VIII B domain with the human factor V Bdomain has been performed (U.S. Pat. No. 5,004,803, incorporated hereinby reference in its entirety). The cDNA sequence encoding human factorVIII and predicted amino acid sequence are shown in SEQ ID NOs:1 and 2,respectively, of US Application Publ. No. 2005/0100990, incorporatedherein by reference in its entirety.

U.S. Pat. No. 5,859,204, Lollar, J. S., incorporated herein by referencein its entirety, reports functional mutants of factor VIII havingreduced antigenicity and reduced immunoreactivity. U.S. Pat. No.6,376,463, Lollar, J. S., incorporated herein by reference in itsentirety, also reports mutants of factor VIII having reducedimmunoreactivity. US Application Publ. No. 2005/0100990, Saenko et al.,incorporated herein by reference in its entirety, reports functionalmutations in the A2 domain of factor VIII.

A number of functional factor VIII molecules, including B-domaindeletions, are disclosed in the following patents U.S. Pat. No.6,316,226 and U.S. Pat. No. 6,346,513, both assigned to Baxter; U.S.Pat. No. 7,041,635 assigned to In2Gen; U.S. Pat. No. 5,789,203, U.S.Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620assigned to Chiron; U.S. Pat. No. 5,972,885 and U.S. Pat. No. 6,048,720assigned to Biovitrum, U.S. Pat. No. 5,543,502 and U.S. Pat. No.5,610,278 assigned to Novo Nordisk; U.S. Pat. No. 5,171,844 assigned toImmuno Ag; U.S. Pat. No. 5,112,950 assigned to Transgene S.A.; U.S. Pat.No. 4,868,112 assigned to Genetics Institute, each of which isincorporated herein by reference in its entirety.

The porcine factor VIII sequence is published, (Toole, J. J., et al.,Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)), incorporated herein byreference in its entirety, and the complete porcine cDNA sequenceobtained from PCR amplification of factor VIII sequences from a pigspleen cDNA library has been reported (Healey, J. F. et al., Blood88:4209-4214 (1996), incorporated herein by reference in its entirety).Hybrid human/porcine factor VIII having substitutions of all domains,all subunits, and specific amino acid sequences were disclosed in U.S.Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093, incorporatedherein by reference in its entirety. More recently, the nucleotide andcorresponding amino acid sequences of the A1 and A2 domains of porcinefactor VIII and a chimeric factor VIII with porcine A1 and/or A2 domainssubstituted for the corresponding human domains were reported in WO94/11503, incorporated herein by reference in its entirety. U.S. Pat.No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA anddeduced amino acid sequences. U.S. Pat. No. 6,458,563, incorporatedherein by reference in its entirety assigned to Emory discloses aB-domain deleted porcine Factor VIII.

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) maybe at least 90% or 95% identical to a Factor VIII amino acid sequenceshown in Table 2 without a signal sequence (amino acids 20 to 1457 ofSEQ ID NO:2; and amino acids 20 to 2351 of SEQ ID NO:6), wherein saidFactor VIII portion has Factor VIII activity. The Factor VIII (or FactorVIII portion of a chimeric polypeptide) may be identical to a FactorVIII amino acid sequence shown in Table 2 without a signal sequence(amino acids 20 to 1457 of SEQ ID NO:2; and amino acids 20 to 2351 ofSEQ ID NO:6).

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) maybe at least 90% or 95% identical to a Factor VIII amino acid sequenceshown in Table 2 with a signal sequence (amino acids 1 to 1457 of SEQ IDNO:2 and amino acids 1 to 2351 of SEQ ID NO:6), wherein said Factor VIIIportion has Factor VIII activity. The Factor VIII (or Factor VIIIportion of a chimeric polypeptide) may be identical to a Factor VIIIamino acid sequence shown in Table 2 with a signal sequence (amino acids1 to 1457 of SEQ ID NO:2 and amino acids 1 to 2351 of SEQ ID NO:6).

“Equivalent dose,” as used herein, means the same dose of Factor VIIIactivity as expressed in International Units, which is independent ofmolecular weight of the polypeptide in question. One International Unit(IU) of factor VIII activity corresponds approximately to the quantityof factor VIII in one milliliter of normal human plasma. Several assaysare available for measuring Factor VIII activity, including the EuropeanPharmacopoeia chromogenic substrate assay and a one stage clottingassay.

“Fc,” as used herein, means functional neonatal Fc receptor (FcRn)binding partners, unless otherwise specified. An FcRn binding partner isany molecule that can be specifically bound by the FcRn receptor withconsequent active transport by the FcRn receptor of the FcRn bindingpartner. Thus, the term Fc includes any variants of IgG Fc that arefunctional. The region of the Fc portion of IgG that binds to the FcRnreceptor has been described based on X-ray crystallography (Burmeisteret al., Nature 372:379 (1994), incorporated herein by reference in itsentirety). The major contact area of the Fc with the FcRn is near thejunction of the CH2 and CH3 domains. Fc-FcRn contacts are all within asingle Ig heavy chain. The FcRn binding partners include, e.g., wholeIgG, the Fc fragment of IgG, and other fragments of IgG that include thecomplete binding region of FcRn. The major contact sites include aminoacid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 ofthe CH2 domain and amino acid residues 385-387, 428, and 433-436 of theCH3 domain. References made to amino acid numbering of immunoglobulinsor immunoglobulin fragments, or regions, are all based on Kabat et al.1991, Sequences of Proteins of Immunological Interest, U. S. Departmentof Public Health, Bethesda; MD, incorporated herein by reference in itsentirety. (The FcRn receptor has been isolated from several mammalianspecies including humans. The sequences of the human FcRn, rat FcRn, andmouse FcRn are known (Story et al., J. Exp. Med. 180: 2377 (1994),incorporated herein by reference in its entirety.) An Fc may comprisethe CH2 and CH3 domains of an immunoglobulin with or without the hingeregion of the immunoglobulin. Exemplary Fc variants are provided in WO2004/101740 and WO 2006/074199, incorporated herein by reference in itsentirety.

Fc (or Fc portion of a chimeric polypeptide) may contain one or moremutations, and combinations of mutations.

Fc (or Fc portion of a chimeric polypeptide) may contain mutationsconferring increased half-life such as M252Y, S254T, T256E, andcombinations thereof, as disclosed in Oganesyan et al., Mol. Immunol.46:1750 (2009), which is incorporated herein by reference in itsentirety; H433K, N434F, and combinations thereof, as disclosed inVaccaro et al., Nat. Biotechnol. 23:1283 (2005), which is incorporatedherein by reference in its entirety; the mutants disclosed at pages 1-2,paragraph [0012], and Examples 9 and 10 of US 2009/0264627 A1, which isincorporated herein by reference in its entirety; and the mutantsdisclosed at page 2, paragraphs [0014] to [0021] of US 20090163699 A1,which is incorporated herein by reference in its entirety.

Fc (or Fc portion of a chimeric polypeptide) may also include, e.g., thefollowing mutations: The Fc region of IgG can be modified according towell recognized procedures such as site directed mutagenesis and thelike to yield modified IgG or Fc fragments or portions thereof that willbe bound by FcRn. Such modifications include, e.g., modifications remotefrom the FcRn contact sites as well as modifications within the contactsites that preserve or even enhance binding to the FcRn. For example thefollowing single amino acid residues in human IgG1 Fc (Fcy1) can besubstituted without significant loss of Fc binding affinity for FcRn:P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A,S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A,E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F,N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A,N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q,A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A,R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A,Y373A, S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A,N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A,Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A,S440A, S444A, and K447A, where for example P238A represents wildtypeproline substituted by alanine at position number 238. In addition toalanine other amino acids may be substituted for the wildtype aminoacids at the positions specified above. Mutations may be introducedsingly into Fc giving rise to more than one hundred FcRn bindingpartners distinct from native Fc. Additionally, combinations of two,three, or more of these individual mutations may be introduced together,giving rise to hundreds more FcRn binding partners. Certain of thesemutations may confer new functionality upon the FcRn binding partner.For example, one embodiment incorporates N297A, removing a highlyconserved N-glycosylation site. The effect of this mutation is to reduceimmunogenicity, thereby enhancing circulating half-life of the FcRnbinding partner, and to render the FcRn binding partner incapable ofbinding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromisingaffinity for FcRn (Routledge et al. 1995, Transplantation 60:847, whichis incorporated herein by reference in its entirety; Friend et al. 1999,Transplantation 68:1632, which is incorporated herein by reference inits entirety; Shields et al. 1995, J. Biol. Chem. 276:6591, which isincorporated herein by reference in its entirety). Additionally, atleast three human Fc gamma receptors appear to recognize a binding siteon IgG within the lower hinge region, generally amino acids 234-237.Therefore, another example of new functionality and potential decreasedimmunogenicity may arise from mutations of this region, as for exampleby replacing amino acids 233-236 of human IgG1 “ELLG” to thecorresponding sequence from IgG2 “PVA” (with one amino acid deletion).It has been shown that FcyRI, FcyRII, and FcyRIII which mediate variouseffector functions will not bind to IgG1 when such mutations have beenintroduced (Ward and Ghetie, Therapeutic Immunology 2:77 (1995), whichis incorporated herein by reference in its entirety; and Armour et al.,Eur. J. Immunol. 29:2613 (1999), which is incorporated herein byreference in its entirety). As a further example of new functionalityarising from mutations described above affinity for FcRn may beincreased beyond that of wild type in some instances. This increasedaffinity may reflect an increased “on” rate, a decreased “off” rate orboth an increased “on” rate and a decreased “off” rate. Mutationsbelieved to impart an increased affinity for FcRn include, e.g., T256A,T307A, E380A, and N434A (Shields et al., J. Biol. Chem. 276:6591 (2001),which is incorporated herein by reference in its entirety).

The Fc (or Fc portion of a chimeric polypeptide) may be at least 90% or95% identical to the Fc amino acid sequence shown in Table 2 (aminoacids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ IDNO:6). The Fc (or Fc portion of a chimeric polypeptide) may be identicalto the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684of SEQ ID NO:2 and amino acids 2352 to 2578 of SEQ ID NO:6).

“Hybrid” polypeptides and proteins, as used herein, means a combinationof a chimeric polypeptide with a second polypeptide. The chimericpolypeptide and the second polypeptide in a hybrid may be associatedwith each other via protein-protein interactions, such as charge-chargeor hydrophobic interactions. The chimeric polypeptide and the secondpolypeptide in a hybrid may be associated with each other via disulfideor other covalent bond(s). Hybrids are described in WO 2004/101740 andWO 2006/074199, each of which is incorporated herein by reference in itsentirety. See also U.S. Pat. Nos. 7,404,956 and 7,348,004, each of whichis incorporated herein by reference in its entirety. The secondpolypeptide may be a second copy of the same chimeric polypeptide or itmay be a non-identical chimeric polypeptide. See, e.g., FIG. 1, Example1, and Table 2. In one embodiment, the second polypeptide is apolypeptide comprising an Fc. In another embodiment, the chimericpolypeptide is a chimeric Factor VIII-Fc polypeptide and the secondpolypeptide consists essentially of Fc, e.g., the hybrid polypeptide ofExample 1, which is a rFVIIIFc recombinant fusion protein consisting ofa single molecule of recombinant B-domain deleted human FVIII(BDD-rFVIII) fused to the dimeric Fc domain of the human IgG1, with nointervening linker sequence. This hybrid polypeptide is referred toherein as FVIIIFc monomeric Fc fusion protein, FVIIIFc monomer hybrid,monomeric FVIIIIFc hybrid, and FVIIIFc monomer-dimer See Example 1, FIG.1, and Table 2A. The Examples provide preclinical and clinical data forthis hybrid polypeptide.

The second polypeptide in a hybrid may comprise or consist essentiallyof a sequence at least 90% or 95% identical to the amino acid sequenceshown in Table 2A(ii) without a signal sequence (amino acids 21 to 247of SEQ ID NO:4) or at least 90% or 95% identical to the amino acidsequence shown in Table 2A(ii) with a signal sequence (amino acids 1 to247 of SEQ ID NO:4). The second polypeptide may comprise or consistessentially of a sequence identical to the amino acid sequence shown inTable 2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ IDNO:4) or identical to the amino acid sequence shown in Table 2A(ii) witha signal sequence (amino acids 1 to 247 of SEQ ID NO:4).

FIG. 1 is a schematic showing the structure of a B domain deleted factorVIII-Fc chimeric polypeptide, and its association with a secondpolypeptide that is an Fc polypeptide. To obtain this hybrid, the codingsequence of human recombinant B-domain deleted FVIII was obtained byreverse transcription-polymerase chain reaction (RT-PCR) from humanliver poly A RNA (Clontech) using FVIII-specific primers. The FVIIIsequence includes the native signal sequence for FVIII. The B-domaindeletion was from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638;4969 bp) for a total deletion of 2682 bp. Then, the coding sequence forhuman recombinant Fc was obtained by RT-PCR from a human leukocyte cDNAlibrary (Clontech) using Fc specific primers. Primers were designed suchthat the B-domain deleted FVIII sequence was fused directly to theN-terminus of the Fc sequence with no intervening linker. The FVIIIFcDNA sequence was cloned into the mammalian dual expression vectorpBUDCE4.1 (Invitrogen) under control of the CMV promoter. A secondidentical Fc sequence including the mouse Igk signal sequence wasobtained by RT-PCR and cloned downstream of the second promoter, EF1α,in the expression vector pBUDCE4.1.

The rFVIIIFc expression vector was transfected into human embryonickidney 293 cells (HEK293H; Invitrogen) using Lipofectamine 2000transfection reagent (Invitrogen). Stable clonal cell lines weregenerated by selection with Zeocin (Invitrogen). One clonal cell line,3C4-22 was used to generate FVIIIFc for characterization in vivo.Recombinant FVIIIFc was produced and purified (McCue et al. 2009) atBiogen Idec (Cambridge, Mass.). The transfection strategy describedabove was expected to yield three products, i.e., monomeric rFVIIIFchybrids, dimeric rFVIIIFc hybrids and dimeric Fc. However, there wasessentially no dimeric rFVIIIFc detected in the conditioned medium fromthese cells. Rather, the conditioned medium contained Fc and monomericrFVIIIFc. It is possible that the size of dimeric rFVIIIFc was too greatand prevented efficient secretion from the cell. This result wasbeneficial since it rendered the purification of the monomer lesscomplicated than if all three proteins had been present. The materialused in these studies had a specific activity of approximately 9000IU/mg.

In one embodiment, the polypeptides of the invention are administered toa patient who expresses high level of von Willebrand factor (VWF).“Subject” or “patient” as used herein means a human individual. Asubject can be a patient who is currently suffering from a bleedingdisorder or is expected to be in need of such a treatment. “Subject” caninclude an adult or a pediatric subject. The pediatric subject can be apediatric patient under the age of 12. The term “pediatrics” as usedherein is the branch of medicine that deals with the care of infants andchildren and the treatment of their diseases. In one embodiment, thesubject is a pediatric patient who has a diagnosis of severe hemophiliaA. In certain embodiments, pediatric subjects are treated with along-acting Factor VIII polypeptide of the invention.

VWF is a plasma protein having a multimer structure in which themolecular weight of the various forms varies between approximately 230kDa for each monomer subunit and up to more than 20 million Da in themultimer forms of greater molecular weight, thus forming the largestknown soluble protein. Its plasma concentration is approximately around5-10 μg/ml (Siedlecki et al., Blood, vol 88: 2939-2950 (1996)) and theplasma form of smaller size is that corresponding to the dimer, with anapproximate size of 500 kDa.

VWF has an essential role to play in primary haemostasis, beingresponsible for the adhesion of platelets to damaged vascular surfacesand therefore formation of the platelet plug on which the mechanisms forformation of the fibrin coagulate develop. It is suggested that thehigher molecular weight multimers support platelet adhesion mechanismsto the sub-endothelium with greater efficiency and the clinical efficacyof VWF concentrates has been related to the concentration of thesemultimers of higher molecular weight (Metzner et al., Haemophilia4:25-32 (1998).

Therefore, subjects expressing high levels of VWF would require lessfrequent dosing of FVIII compared to a subject who expresses lower ornormal levels of VWF. The average range of VWF in plasma is betweenabout 50 IU/dL and about 200 IU/dL. In one embodiment, the average levelof VWF in plasma is about 50 IU/dL. In another embodiment, a VWF levelin plasma of at least about 100 IU/dL is considered a high VWF level. Inanother embodiment, a high level of VWF in plasma is between about 100IU/dL and about 200 IU/dL. In another embodiment a high level of VWF inplasma is at least about 110 IU/dL, about 120 IU/dL, about 130 IU/dL,about 140 IU/dL, about 150 IU/dL, about 160 IU/dL, about 170 IU/dL,about 180 IU/dL, about 190 IU/dL, or about 200 IU/dL.

Therefore, in one embodiment, subjects expressing at least about 100IU/dL of plasma VWF are administered a long-acting FVIII polypeptide ofthe invention at a long interval dosing regimen. In one embodiment, thelong-acting FVIII polypeptide is administered at a dosing interval of atleast about 3 days. In another embodiment, the long-acting FVIIIpolypeptide is administered at a dosing interval of at least about onceevery week, about once every two weeks, about once every 15 days, aboutonce every 20 days, about once every three weeks, about once every 25days, about once every four weeks, or about once every one month.

In one embodiment, the subjects were previously identified as havinghigh levels of VWF. In certain embodiments, subjects having a bloodserotype other than O (i.e., A, B, or AB) require less frequent dosingof long-acting FVIII because the long-acting FVIII has a longerhalf-life in these subjects. In these subjects, the increased half-lifeis due to their elevated VWF levels.

Moreover, pharmacokinetic data, defined as the study of the time courseof drug absorption, distribution, metabolism, and excretion, can be usedas an identifier of subjects eligible for longer or shorter dosingintervals using a long-acting FVIII polypeptide of the invention.Cinical pharmacokinetics is the application of pharmacokineticprinciples to the safe and effective therapeutic management of drugs inan individual patient. The primary goals of clinical pharmacokineticsinclude enhancing efficacy and decreasing toxicity of a patient's drugtherapy. The development of strong correlations between drugconcentrations and their pharmacologic responses has enabled cliniciansto apply pharmacokinetic principles to actual patient situations.

Thus, in one embodiment, the half-life of a FVIII-Fc polypeptide of theinvention is used to identify patients who express high levels of VWF.The range of half-life of FVIII-Fc is between about 10 and about 40hours, depending at least in part on the levels of VWF also present. Onaverage however, the half-life of FVIII-Fc is about 18 hours. Generally,FVIII-Fc exhibits an increased half-life of at least about 1.2-fold inpatients having high levels of VWF compared to the half-life of FVIII-Fcwhen administered to individuals having average levels of VWF. In oneembodiment, FVIII-Fc exhibits an increased half-life of at least about1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-foldcompared to the half-life of FVIII-Fc when administered to individualshaving average levels of VWF. In one embodiment, in subjects expressinghigh levels of VWF, the half-life of FVIII-Fc is between at least about20 hours and about 40 hours. In another embodiment, the half-life ofFVIII-Fc is at least about 21 hours, 22 hours, 23 hours, 24 hours, 25hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39hours, or 40 hours. In one embodiment the half-life of FVIII-Fc isbetween about 20 and about 27 hours in subjects having high levels ofVWF. Thus, in one embodiment, an increased half-life of FVIII-Fccompared to average values is indicative of a subject that is eligiblefor a longer dosing interval with a long-acting FVIII polypeptide of theinvention.

In another embodiment, the half-life of a short-acting FVIII polypeptideis used to identify patients who express high levels of VWF. As usedherein, the term “short-acting FVIII” refers to a FVIII polypeptide inwhich no extenders of half-life have been added. In one embodiment,short-acting FVIII polypeptides consist of full-length or Bdomain-deleted FVIII. Examples of short-acting FVIII polypeptides areAdvate® and ReFacto®.

Since the half-life of short-acting FVIII also varies depending at leastin part on VWF levels, short-acting FVIII polypeptides can also be usedto identify patients that are eligible for a longer dosing interval of along-acting FVIII polypeptide of the invention. In one embodiment, theshort-acting FVIII exhibits an increased half-life of at least about1.2-fold in individuals expressing high levels of VWF compared to thehalf-life of the short-acting FVIII when administered to individualshaving average levels of VWF. In another embodiment, the short-actingFVIII exhibits an increased half-life of at least about 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold in individualsexpressing high levels of VWF compared to the half-life of theshort-acting FVIII when administered to individuals having averagelevels of VWF. Thus, individuals that demonstrate an increased half-lifeof at least about 1.2-fold when they are administered a short-actingFVIII are eligible for a longer dosing interval with a long-acting FVIIIpolypeptide of the invention.

“Dosing interval,” as used herein, means the dose of time that elapsesbetween multiple doses being administered to a subject. The comparisonof dosing interval may be carried out in a single subject or in apopulation of subjects and then the average obtained in the populationmay be calculated.

The dosing interval when administering a chimeric Factor VIIIpolypeptide, e.g., a chimeric Factor VIII-Fc polypeptide (a polypeptidecomprising a Factor VIII or a hybrid) of the invention may be at leastabout one and one-half times longer than the dosing interval requiredfor an equivalent dose of said Factor VIII without the non-Factor VIIIportion, e.g., without the Fc portion (a polypeptide consisting of saidFactor VIII). The dosing interval may be at least about one and one-halfto six times longer, one and one-half to five times longer, one andone-half to four times longer, one and one-half to three times longer,or one and one-half to two times longer, than the dosing intervalrequired for an equivalent dose of said Factor VIII without thenon-Factor VIII portion, e.g., without the Fc portion (a polypeptideconsisting of said Factor VIII). The dosing interval may be at leastabout one and one-half, two, two and one-half, three, three andone-half, four, four and one-half, five, five and one-half or six timeslonger than the dosing interval required for an equivalent dose of saidFactor VIII without the non-Factor VIII portion, e.g., without the Fcportion (a polypeptide consisting of said Factor VIII). The dosinginterval may be about every three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen days or longer. The dosinginterval may be at least about one and one-half to 5, one and one-half,2, 3, 4, or 5 days or longer. For on-demand treatment, the dosinginterval of said chimeric polypeptide or hybrid is about once every24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, or 72 hours or longer.

In one embodiment, the effective dose is 25-80 IU/kg (25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 62, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 IU/kg)and the dosing interval is once every 3-5, 3-6, 3-7, 3, 4, 5, 6, 7, or 8or more days, or three times per week, or no more than three times perweek. In one embodiment, the effective dose is 80 IU/kg and the dosinginterval is once every 3 days. In a further embodiment, the effectivedose of 80 IU/kg given at a dosing interval of every 3 days isadministered to a pediatric subject. In another embodiment, theeffective dose is 65 IU/kg and the dosing interval is once weekly, oronce every 6-7 days. The doses can be administered repeatedly as long asthey are necessary (e.g., at least 10, 20, 28, 30, 40, 50, 52, or 57weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years).

In certain embodiments, the effective dose for on-demand treatment is20-50 IU/Kg (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50IU/kg). The on-demand treatment can be one time dosing or repeateddosing. For repeated dosing, the dosing interval can be every 12-24hours, every 24-36 hours, every 24-48 hours, every 36-48 hours, or every48-72 hours.

“Long-acting Factor VIII” is a Factor VIII having an increased half-life(also referred to herein as t1/2, t1/2 beta, elimination half-life andHL) over a reference Factor VIII. The increased half-life of along-acting Factor VIII may be due to fusion to one or more non-FactorVIII polypeptides such as, e.g., Fc, XTEN, albumin, a PAS sequence,transferrin, CTP (28 amino acid C-terminal peptide (CTP) of hCG with its4 O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES),albumin binding polypeptide, albumin-binding small molecules, or two ormore combinations thereof. The increased half-life may be due to one ormore modification, such as, e.g., pegylation. Exemplary long-actingFactor VIII polypeptides include, e.g., chimeric Factor VIIIpolypeptides comprising Fc, chimeric Factor VIII polypeptides comprisingXTEN and chimeric Factor VIII polypeptides comprising albumin.Additional exemplary long-acting Factor VIII polypeptides include, e.g.,pegylated Factor VIII.

The “reference” polypeptide, in the case of a long-acting chimericFactor VIII polypeptide, is a polypeptide consisting essentially of theFactor VIII portion of the chimeric polypeptide, e.g., the same FactorVIII portion without the Fc portion, without the XTEN portion, orwithout the albumin portion. Likewise, the reference polypeptide in thecase of a modified Factor VIII is the same Factor VIII without themodification, e.g., a Factor VIII without the pegylation.

In some embodiments, the long-acting Factor VIII has one or more of thefollowing properties when administered to a subject:

a mean residence time (MRT) (activity) in said subject of about 14-41.3hours;a clearance (CL) (activity) in said subject of about 1.22-5.19mL/hour/kg or less;a t1/2beta (activity) in said subject of about 11-26.4 hours;an incremental recovery (K value) (activity; observed) in said subjectof about 1.38-2.88 IU/dL per IU/kg;a Vss (activity) in said subject of about 37.7-79.4 mL/kg; andan AUC/dose in said subject of about 19.2-81.7 IU*h/dL per IU/kg.

In some embodiments, the long-acting Factor VIII has one or more of thefollowing properties when administered to a patient population:

a mean incremental recovery (K-Value) (activity; observed) greater that1.38 IU/dL per IU/kg;a mean incremental recovery (K-Value) (activity; observed) of at leastabout 1.5, at least about 1.85, or at least about 2.46 IU/dL per IU/kg.a mean clearance (CL) (activity) in said patient population of about2.33±1.08 mL/hour/kg or less;a mean clearance (CL) (activity) in said patient population of about1.8-2.69 mL/hour/kg;a mean clearance (CL) (activity) in said patient population that isabout 65% of the clearance of a polypeptide comprising said Factor VIIIwithout modification;a mean mean residence time (MRT) (activity) in said patient populationof at least about 26.3±8.33 hours;a mean MRT (activity) in said patient population of about 25.9-26.5hours;a mean MRT (activity) in said patent population that is about 1.5 foldlonger than the mean MRT of a polypeptide comprising said Factor VIIIwithout modification;a mean t1/2beta (activity) in said patient population of about 18.3±5.79hours;a mean t1/2beta (activity) in said patient population that is about18-18.4 hours;a mean t1/2beta (activity) in said patient population that is about 1.5fold longer than the mean t1/2beta of a polypeptide comprising saidFactor VIII without modification;a mean incremental recovery (K value) (activity; observed) in saidpatient population of about 2.01±0.44 IU/dL per IU/kg;a mean incremental recovery (K value) (activity; observed) in saidpatient population of about 1.85-2.46 IU/dL per IU/kg;a mean incremental recovery (K value) (activity; observed) in saidpatient population that is about 90% of the mean incremental recovery ofa polypeptide comprising said Factor VIII without modification;a mean Vss (activity) in said patient population of about 55.1±12 3mL/kg;a mean Vss (activity) in said patient population of about 45.3-56.1mL/kg;a mean AUC/dose (activity) in said patient population of about 49.9±18.2IU*h/dL per IU/kg;a mean AUC/dose (activity) in said patient population of about 44.8-57.6IU*h/dL per IU/kg.

In other embodiments, the long-acting Factor VIII has one or more of thefollowing properties when administered to a patient population:

a C_(max) _(—) OBS in said subject administered with the chimericpolypeptide is comparable to the C_(max) _(—) OBS in a subjectadministered with the same amount of a polypeptide consisting of thefull-length, mature Factor VIII when measured by a one stage (aPTT)assay or a two stage (chromogenic) assay;

a C_(max) _(—) OBS in said subject of about 60.5 IU/dL, about 60.5±1IU/dL, about 60.5±2 IU/dL, about 60.5±3 IU/dL, about 60.5±4 IU/dL, about60.5±5 IU/dL, about 60.5±6 IU/dL, about 60.5±7 IU/dL, about 60.5±8IU/dL, about 60.5±9 IU/dL, or about 60.5±10 IU/dL as measured by a onestage (aPTT) assay when about 25 IU/kg of the chimeric polypeptide isadministered;

a C_(max) _(—) OBS in said subject of about 53.1-69 IU/dL as measured bya one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptideis administered;

a C_(max) _(—) OBS in said subject of about 119 IU/dL, about 119±1IU/dL, about 119±2 IU/dL, about 119±3 IU/dL, about 119±4 IU/dL, about119±5 IU/dL, about 119±6 IU/dL, about 119±7 IU/dL, about 119±8 IU/dL,about 119±9 IU/dL, about 119±10 IU/dL, about 119±11 IU/dL, about 119±12IU/dL, about 119±13 IU/dL, about 119±14 IU/dL, about 119±15 IU/dL, about119±16 IU/dL, about 119±17 IU/dL, or about 119±18 IU/dL, as measured bya one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptideis administered;

a C_(max) _(—) OBS in said subject of about 103-136 IU/dL as measured bya one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptideis administered;

a C_(max) _(—) OBS in said subject of about 76.5 IU/dL, about 76.5±1IU/dL, about 76.5±2 IU/dL, about 76.5±3 IU/dL, about 76.5±4 IU/dL, about76.5±5 IU/dL, about 76.5±6 IU/dL, about 76.5±7 IU/dL, about 76.5±8IU/dL, about 76.5±9 IU/dL, about 76.5±10 IU/dL, about 76.5±11 IU/dL,about 76.5±12 IU/dL, about 76.5±13 IU/dL, about 76.5±14 IU/dL, or about76.5±15 IU/dL, as measured by a two stage (chromogenic) assay when about25 IU/kg of the chimeric polypeptide is administered;

a C_(max) _(—) OBS in said subject of about 64.9-90.1 IU/dL as measuredby a two stage (chromogenic) assay when about 25 IU/kg of the chimericpolypeptide is administered;

a C_(max) _(—) OBS in said subject of about 182 IU/dL, about 182±2IU/dL, about 182±4 IU/dL, about 182±6 IU/dL, about 182±8 IU/dL, about182±10 IU/dL, about 182±12 IU/dL, about 182±14 IU/dL, about 182±16IU/dL, about 182±18 IU/dL, or about 182±20 IU/dL as measured by a twostage (chromogenic) assay when about 65 IU/kg of the chimericpolypeptide is administered; or

a C_(max) _(—) OBS in said subject of about 146-227 IU/dL, about 146±5IU/dL, about 146±10 IU/dL, about 227±5 IU/dL, or about 146±10 IU/dL asmeasured by a two stage (chromogenic) assay when about 65 IU/kg of thechimeric polypeptide is administered.

In certain embodiments, the long-acting Factor VIII has one or more ofthe following properties when administered to a patient population:

a t1/2beta (activity) in said subject that is at least 1.48, 1.49, 1.50,1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62,1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74,1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86,1.87, 1.88, 1.89, or 1.90 times higher than the t1/2beta (activity) in asubject administered with the same amount of a polypeptide consisting ofthe full-length, mature Factor VIII when measured by a one stage (aPTT)assay or a two stage (chromogenic) assay;

a t1/2beta (activity) in said subject of about 18.8 hours, 18.8±1 hours,18.8±1 hours, 18.8±2 hours, 18.8±3 hours, 18.8±4 hours, 18.8±5 hours,18.8±6 hours, 18.8±7 hours, 18.8±8 hours, 18.8±9 hours, 18.8±10 hours,or 18.8±11 hours as measured by a one stage (aPTT) assay;

a t1/2beta (activity) in said subject of about 14.3-24.5 hours asmeasured by a one stage (aPTT) assay;

a t1/2beta (activity) in said subject of about 16.7 hours, 16.7±1 hours,16.7±2 hours, 16.7±3 hours, 16.7±4 hours, 16.7±5 hours, 16.7±6 hours,16.7±7 hours, 16.7±8 hours, 16.7±9 hours, 16.7±10 hours, or 16.7±11hours as measured by a two stage (chromogenic) assay;

a t1/2beta (activity) in said subject of about 13.8-20.1 hours asmeasured by a two stage (chromogenic) assay;

a t1/2beta (activity) in said subject of about 19.8 hours, 19.8±1 hours,19.8±2 hours, 19.8±3 hours, 19.8±4 hours, 19.8±5 hours, 19.8±6 hours,19.8±7 hours, 19.8±8 hours, 19.8±9 hours, 19.8±10 hours, or 19.8±11hours as measured by a two stage (chromogenic) assay; or

a t1/2beta (activity) in said subject of about 14.3-27.5 hours asmeasured by a two stage (chromogenic) assay.

In certain embodiments, the long-acting Factor VIII has one or more ofthe following properties when administered to a patient population:

a clearance (CL) (activity) in said subject is 0.51, 0.52, 0.53, 0.54,0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66,0.67, 0.68, 0.69, or 0.70 times lower than the clearance in a subjectadministered with the same amount of a polypeptide consisting of thefull-length, mature Factor VIII when measured by a one stage (aPTT)assay or a two stage (chromogenic) assay;

a clearance (CL) (activity) in said subject of about 1.68 mL/hour/kg,1.68±0.1 mL/hour/kg, 1.68±0.2 mL/hour/kg, 1.68±0.3 mL/hour/kg, 1.68±0.4mL/hour/kg, 1.68±0.5 mL/hour/kg, 1.68±0.6 mL/hour/kg, or 1.68±0 7mL/hour/kg, as measured by a one stage (aPTT) assay when about 25 IU/kgof the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.31-2.15mL/hour/kg as measured by a one stage (aPTT) assay when about 25 IU/kgof the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 2.32 mL/hour/kg,2.32±0.1 mL/hour/kg, 2.32±0.2 mL/hour/kg, 2.32±0.3 mL/hour/kg, 2.32±0.4mL/hour/kg, 2.32±0.5 mL/hour/kg, 2.32±0.6 mL/hour/kg, or 2.32±0 7mL/hour/kg as measured by a one stage (aPTT) assay when about 65 IU/kgof the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.64-3.29mL/hour/kg as measured by oa ne stage (aPTT) assay when about 65 IU/kgof the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.49 mL/hour/kg,1.49±0.1 mL/hour/kg, 1.49±0.2 mL/hour/kg, 1.49±0.3 mL/hour/kg, 1.49±0.4mL/hour/kg, 1.49±0.5 mL/hour/kg, 1.49±0.6 mL/hour/kg, or 1.49±0 7mL/hour/kg as measured by a two stage (chromogenic) assay when about 25IU/kg of the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.16-1.92mL/hour/kg as measured by a two stage (chromogenic) assay when about 25IU/kg of the chimeric polypeptide is administered;

a clearance (CL) (activity) in said subject of about 1.52 mL/hour/kg,1.52±0.1 mL/hour/kg, 1.52±0.2 mL/hour/kg, 1.52±0.3 mL/hour/kg, 1.52±0.4mL/hour/kg, 1.52±0.5 mL/hour/kg, 1.52±0.6 mL/hour/kg, or 1.52±0 7mL/hour/kg as measured by a two stage (chromogenic) assay when about 65IU/kg of the chimeric polypeptide is administered; or

a clearance (CL) (activity) in said subject of about 1.05-2.20mL/hour/kg as measured by a two stage (chromogenic) assay when about 65IU/kg of the chimeric polypeptide is administered.

In some embodiments, the long-acting Factor VIII has one or more of thefollowing properties when administered to a patient population:

a MRT in said subject is at least 1.46, 1.47, 1.48, 1.49, 1.50, 1.51,1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63,1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75,1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87,1.88, 1.89, 1.90, 1.91, 1.92, or 1.93 times higher than the MRT in asubject administered with the same amount of a polypeptide consisting ofthe full-length, mature Factor VIII when meansured by a one stage (aPTT)assay or a two stage (chromogenic) assay;

a MRT (activity) in said subject of about 27 hours, 27±1 hours, 27±2hours, 27±3 hours, 27±4 hours, 27±5 hours, 27±6 hours, 27±7 hours, 27±8hours, 27±9 hours, or 27±10 hours as meansured by a one stage (aPTT)assay;

a MRT (activity) in said subject of about 20.6-35.3 hours as meansuredby a one stage (aPTT) assay;

a MRT (activity) in said subject of about 23.9-28.5 hours as measured bya two stage (chromogenic) assay;

a MRT (activity) in said subject of about 19.8-28.9 hours as measured bya two stage (chromogenic) assay; or

a MRT (activity) in said subject of about 20.5-39.6 hours as measured bya two stage (chromogenic) assay.

In other embodiments, the long-acting Factor VIII has one or more of thefollowing properties when administered to a patient population:

an incremental recovery in said subject that is comparable to theIncremental Recovery in a subject administered with the same amount of apolypeptide consisting of the full-length, mature Factor VIII whenmeasured by a one stage (aPTT) assay or a two stage (chromogenic) assay;

an incremental recovery in said subject of about 2.44 IU/dL per IU/kg,2.44±0.1 IU/dL per IU/kg, 2.44±0.2 IU/dL per IU/kg, 2.44±0.3 IU/dL perIU/kg, 2.44±0.4 IU/dL per IU/kg, 2.44±0.5 IU/dL per IU/kg, 2.44±0.6IU/dL per IU/kg, 2.44±0.7 IU/dL per IU/kg, 2.44±0.8 IU/dL per IU/kg,2.44±0.9 IU/dL per IU/kg, 2.44±1.0 IU/dL per IU/kg, 2.44±1.1 IU/dL perIU/kg, or 2.44±1.2 IU/dL per IU/kg as measured by a one stage (aPTT)assay when about 25 IU/kg of the chimeric polypeptide is administered;

an incremental recovery in said subject of about 2.12-2.81 IU/dL perIU/kg as measured by a one stage (aPTT) assay when about 25 IU/kg of thechimeric polypeptide is administered;

an incremental recovery in said subject of about 1.83 IU/dL per IU/kg,1.83±0.1 IU/dL per IU/kg, 1.83±0.2 IU/dL per IU/kg, 1.83±0.3 IU/dL perIU/kg, 1.83±0.4 IU/dL per IU/kg, 1.83±0.5 IU/dL per IU/kg, 1.83±0.6IU/dL per IU/kg, 1.83±0.7 IU/dL per IU/kg, 1.83±0.8 IU/dL per IU/kg,1.83±0.9 IU/dL per IU/kg, 1.83±1.0 IU/dL per IU/kg, or 1.83±1.1 IU/dLper IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg ofthe chimeric polypeptide is administered;

an incremental recovery in said subject of about 1.59-2.10 IU/dL perIU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of thechimeric polypeptide is administered;

an incremental recovery in said subject of about 3.09 IU/dL per IU/kg,3.09±0.1 IU/dL per IU/kg, 3.09±0.2 IU/dL per IU/kg, 3.09±0.3 IU/dL perIU/kg, 3.09±0.4 IU/dL per IU/kg, 3.09±0.5 IU/dL per IU/kg, 3.09±0.6IU/dL per IU/kg, 3.09±0.7 IU/dL per IU/kg, 3.09±0.8 IU/dL per IU/kg,3.09±0.9 IU/dL per IU/kg, 3.09±1.0 IU/dL per IU/kg, 3.09±1.1 IU/dL perIU/kg, 3.09±1.2 IU/dL per IU/kg, or 3.09±1.3 IU/dL per IU/kg, asmeasured by a two stage (chromogenic) assay when about 25 IU/kg of thechimeric polypeptide is administered;

an incremental recovery in said subject of about 2.80 IU/dL per IU/kg,2.80±0.1 IU/dL per IU/kg, 2.80±0.2 IU/dL per IU/kg, 2.80±0.3 IU/dL perIU/kg, 2.80±0.4 IU/dL per IU/kg, 2.80±0.5 IU/dL per IU/kg, 2.80±0.6IU/dL per IU/kg, 2.80±0.7 IU/dL per IU/kg, 2.80±0.8 IU/dL per IU/kg,2.80±0.9 IU/dL per IU/kg, 2.80±1.0 IU/dL per IU/kg, 2.80±1.1 IU/dL perIU/kg, or 2.80±1.2 IU/dL per IU/kg, as measured by a two stage(chromogenic) assay when about 65 IU/kg of the chimeric polypeptide isadministered;

an incremental recovery in said subject of about 2.61-3.66 IU/dL perIU/kg as measured by a two stage (chromogenic) assay when about 25 IU/kgof the chimeric polypeptide is administered; or

an incremental recovery in said subject of about 2.24-3.50 IU/dL perIU/kg as measured by a two stage (chromogenic) assay when about 65 IU/kgof the chimeric polypeptide is administered.

In still other embodiments, the long-acting Factor VIII has one or moreof the following properties when administered to a patient population:

a Vss (activity) in said subject that is comparable to the Vss(activity) in a subject administered with the same amount of apolypeptide consisting of the full-length, mature Factor VIII whenmeasured by a one stage (aPTT) assay or a two stage (chromogenic) assay;

a Vss (activity) in said subject of about 45.5 mL/kg, 45.5±1 mL/kg,45.5±2 mL/kg, 45.5±3 mL/kg, 45.5±4 mL/kg, 45.5±5 mL/kg, 45.5±6 mL/kg,45.5±7 mL/kg, 45.5±8 mL/kg, 45.5±9 mL/kg, 45.5±10 mL/kg, or 45.5±11mL/kg, as measured by a one stage (aPTT) assay when about 25 IU/kg ofthe chimeric polypeptide is administered;

a Vss (activity) in said subject of about 39.3-52.5 mL/kg as measured bya one stage (aPTT) assay when about 25 IU/kg of the chimeric polypeptideis administered;

a Vss (activity) in said subject of about 62.8 mL/kg, 62.8±1 mL/kg,62.8±2 mL/kg, 62.8±3 mL/kg, 62.8±4 mL/kg, 62.8±5 mL/kg, 62.8±6 mL/kg,62.8±7 mL/kg, 62.8±8 mL/kg, 62.8±9 mL/kg, 62.8±10 mL/kg, 62.8±11 mL/kg,62.8±12 mL/kg, 62.8±13 mL/kg, 62.8±14 mL/kg, 62.8±15 mL/kg, or 62.8±16mL/kg as measured by a one stage (aPTT) assay when about 65 IU/kg of thechimeric polypeptide is administered;

a Vss (activity) in said subject of about 55.2-71.5 mL/kg as measured bya one stage (aPTT) assay when about 65 IU/kg of the chimeric polypeptideis administered;

a Vss (activity) in said subject of about 35.9 mL/kg, 35.9±1 mL/kg,35.9±2 mL/kg, 35.9±3 mL/kg, 35.9±4 mL/kg, 35.9±5 mL/kg, 35.9±6 mL/kg,35.9±7 mL/kg, 35.9±8 mL/kg, 35.9±9 mL/kg, 35.9±10 mL/kg, 35.9±11 mL/kg,35.9±12 mL/kg, or 35.9±13 mL/kg, as measured by a two stage(chromogenic) assay when about 25 IU/kg of the chimeric polypeptide isadministered;

a Vss (activity) in said subject of about 30.4-42.3 mL/kg as measured bya two stage (chromogenic) assay when about 25 IU/kg of the chimericpolypeptide is administered;

a Vss (activity) in said subject of about 43.4 mL/kg, 43.4±1 mL/kg,43.4±2 mL/kg, 43.4±3 mL/kg, 43.4±4 mL/kg, 43.4±5 mL/kg, 43.4±6 mL/kg,43.4±7 mL/kg, 43.4±8 mL/kg, 43.4±9 mL/kg, 43.4±10 mL/kg, 43.4±11 mL/kg,43.4±12 mL/kg, 43.4±13 mL/kg, 43.4±14 mL/kg, 43.4±15 mL/kg, or 43.4±16mL/kg, as measured by a two stage (chromogenic) assay when about 65IU/kg of the chimeric polypeptide is administered; or

a Vss (activity) in said subject of about 38.2-49.2 mL/kg as measured bya two stage (chromogenic) assay when about 65 IU/kg of the chimericpolypeptide is administered.

In yet other embodiments, the long-acting Factor VIII has one or more ofthe following properties when administered to a patient population:

an AUC_(INF) in said subject that is at least 1.45 1.46, 1.47, 1.48,1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60,1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72,1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84,1.85, 1.86, 1.87, 1.88, 1.89, 1.90 times higher than the AUC_(INF) in asubject administered with the same amount of a polypeptide consisting ofthe full-length, mature Factor VIII when measured by a one stage (aPTT)assay or a two stage (chromogenic) assay;

an AUC_(INF) in said subject of about 1440±316 hr*IU/dL per IU/kg asmeasured by a one stage (aPTT) assay when about 25 IU/kg of the chimericpolypeptide is administered;

an AUC_(INF) in said subject of about 1160-1880 hr*IU/dL per IU/kg asmeasured by a one stage (aPTT) assay when about 25 IU/kg of the chimericpolypeptide is administered;

an AUC_(INF) in said subject of about 1480 hr*IU/dL per IU/kg, 1480±100hr*IU/dL per IU/kg, 1480±200 hr*IU/dL per IU/kg, 1480±300 hr*IU/dL perIU/kg, 1480±400 hr*IU/dL per IU/kg, 1480±500 hr*IU/dL per IU/kg,1480±600 hr*IU/dL per IU/kg, 1480±700 hr*IU/dL per IU/kg, 1480±800hr*IU/dL per IU/kg, 1480±900 hr*IU/dL per IU/kg, or 1480±1000 hr*IU/dLper IU/kg, as measured by a one stage (aPTT) assay when about 25 IU/kgof the chimeric polypeptide is administered;

an AUC_(INF) in said subject of about 2910±1320 hr*IU/dL per IU/kg asmeasured by a one stage (aPTT) assay when about 65 IU/kg of the chimericpolypeptide is administered;

an AUC_(INF) in said subject of about 1980-3970 hr*IU/dL per IU/kg asmeasured by a one stage (aPTT) assay when about 65 IU/kg of the chimericpolypeptide is administered;

an AUC_(INF) in said subject of about 2800 hr*IU/dL per IU/kg, 2800±100hr*IU/dL per IU/kg, 2800±200 hr*IU/dL per IU/kg, 2800±300 hr*IU/dL perIU/kg, 2800±400 hr*IU/dL per IU/kg, 2800±500 hr*IU/dL per IU/kg,2800±600 hr*IU/dL per IU/kg, 2800±700 hr*IU/dL per IU/kg, 2800±800hr*IU/dL per IU/kg, 2800±900 hr*IU/dL per IU/kg, or 2800±1000 hr*IU/dLper IU/kg as measured by a one stage (aPTT) assay when about 65 IU/kg ofthe chimeric polypeptide is administered;

an AUC_(INF) in said subject of about 1660 hr*IU/dL per IU/kg, 1660±100hr*IU/dL per IU/kg, 1660±200 hr*IU/dL per IU/kg, 1660±300 hr*IU/dL perIU/kg, 1660±400 hr*IU/dL per IU/kg, 1660±500 hr*IU/dL per IU/kg,1660±600 hr*IU/dL per IU/kg, 1660±700 hr*IU/dL per IU/kg, 1660±800hr*IU/dL per IU/kg, 1660±900 hr*IU/dL per IU/kg, or 1660±1000 hr*IU/dLper IU/kg as measured by a two stage (chromogenic) assay when about 25IU/kg of the chimeric polypeptide is administered;

an AUC_(INF) in said subject of about 1300-2120 hr*IU/dL per IU/kg asmeasured by a two stage (chromogenic) assay when about 25 IU/kg of thechimeric polypeptide is administered;

an AUC_(INF) in said subject of about 4280 hr*IU/dL per IU/kg, 4280±100hr*IU/dL per IU/kg, 4280±200 hr*IU/dL per IU/kg, 4280±300 hr*IU/dL perIU/kg, 4280±400 hr*IU/dL per IU/kg, 4280±500 hr*IU/dL per IU/kg,4280±600 hr*IU/dL per IU/kg, 4280±700 hr*IU/dL per IU/kg, 4280±800hr*IU/dL per IU/kg, 4280±900 hr*IU/dL per IU/kg, 4280±1000 hr*IU/dL perIU/kg, 4280±1100 hr*IU/dL per IU/kg, 4280±1200 hr*IU/dL per IU/kg,4280±1300 hr*IU/dL per IU/kg, 4280±1400 hr*IU/dL per IU/kg, 4280±1500hr*IU/dL per IU/kg, or 4280±1600 hr*IU/dL per IU/kg as measured by a twostage (chromogenic) assay when about 65 IU/kg of the chimericpolypeptide is administered; or

an AUC_(INF) in said subject of about 2960-6190 hr*IU/dL per IU/kg asmeasured by a two stage (chromogenic) assay when about 65 IU/kg of thechimeric polypeptide is administered.

“On-demand treatment,” as used herein, means treatment that is intendedto take place over a short course of time and is in response to anexisting condition, such as a bleeding episode, or a perceived need suchas planned surgery. Conditions that may require on-demand treatmentinclude, e.g., a bleeding episode, hemarthrosis, muscle bleed, oralbleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma,trauma capitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, or bleeding in the illiopsoassheath. The subject may be in need of surgical prophylaxis,peri-operative management, or treatment for surgery. Such surgeriesinclude, e.g., minor surgery, major surgery, tooth extraction,tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement,craniotomy, osteosynthesis, trauma surgery, intracranial surgery,intra-abdominal surgery, intrathoracic surgery, or joint replacementsurgery.

In one embodiment, on-demand treatment resolves greater than 80%(greater than 80%, greater than 81%, greater than 82%, greater than 83%,greater than 84%, greater than 85%, greater than 86%, greater than 87%,greater than 88%, greater than 89%, greater than 90%, greater than 91%,greater than 92%, greater than 93%, greater than 94%, greater than 95%,greater than 96%, greater than 97%, greater than 98%, greater than 99%,or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% ofbleeds (e.g., spontaneous bleeds) in a single dose. In anotherembodiment, greater than 80% (greater than 81%, greater than 82%,greater than 83%, greater than 84%, greater than 85%, greater than 86%,greater than 87%, greater than 88%, greater than 89%, greater than 90%,greater than 91%, greater than 92%, greater than 93%, greater than 94%,greater than 95%, greater than 96%, greater than 97%, greater than 98%,or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% ofbleeding episodes are rated excellent or good by physicians afteron-demand treatment. In other embodiments, greater than 5%, (greaterthan 6%, greater than 7%, greater than 8%, greater than 9%, greater than10%, greater than 11%, greater than 12%, greater than 13%, greater than14%, greater than 15%, greater than 16%, greater than 17%, greater than18%, greater than 19%, greater than 20%), or 5-20%, 5-15%, 5-10%,10-20%, or 10-15% of bleeding episodes are rated as fair by physiciansafter on-demand treatment.

“Polypeptide,” “peptide” and “protein” are used interchangeably andrefer to a polymeric compound comprised of covalently linked amino acidresidues.

“Polynucleotide” and “nucleic acid” are used interchangeably and referto a polymeric compound comprised of covalently linked nucleotideresidues. Polynucleotides may be DNA, cDNA, RNA, single stranded, ordouble stranded, vectors, plasmids, phage, or viruses. Polynucleotidesinclude, e.g., those in Table 1, which encode the polypeptides of Table2 (see Table 1). Polynucleotides also include, e.g., fragments of thepolynucleotides of Table 1, e.g., those that encode fragments of thepolypeptides of Table 2, such as the Factor VIII, Fc, signal sequence,6His and other fragments of the polypeptides of Table 2.

“Prophylactic treatment,” as used herein, means administering a FactorVIII polypeptide in multiple doses to a subject over a course of time toincrease the level of Factor VIII activity in a subject's plasma. Theincreased level can be sufficient to decrease the incidence ofspontaneous bleeding or to prevent bleeding, e.g., in the event of anunforeseen injury. During prophylactic treatment, the plasma proteinlevel in the subject may not fall below the baseline level for thatsubject, or below the level of Factor VIII that characterizes severehemophilia (<1 IU/dl [1%]).

In one embodiment, the prophylaxis regimen is “tailored” to theindividual patient, for example, by determining PK data for each patientand administering Factor VIII of the invention at a dosing interval thatmaintains a trough level of 1-3% FVIII activity. Adjustments may be madewhen a subject experiences unacceptable bleeding episodes defined as ≧2spontaneous bleeding episodes over a rolling two-month period. In thiscase, adjustment will target trough levels of 3-5%. In anotherembodiment, prophylactic treatment results in prevention and control ofbleeding, sustained control of bleeding, sustained protection frombleeding, and/or sustained benefit. Prophylaxis, e.g., sustainedprotection can be demonstrated by an increased AUC to last measured timepoint (AUC-LAST) and reduced clearance, resulting in increased terminalt1/2 compared to short acting FVIII. Prophylaxis can be demonstrated bybetter Cmax, better Tmax, and/or greater mean residence time versusshort-acting FVIII. In some embodiments, prophylaxis results in nospontaneous bleeding episodes within about 24, 36, 48, 72, or 96 hours(e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, 90, 91, 92, 93, 94,95, or 96 hours), after injection (e.g., the last injection). In certainembodiments, prophylaxis results in greater than 30% (e.g., greater than31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 96, 87, 88, 89, or 90%, for example, greater than 50%), meanreduction in annualized bleeding episodes with once weekly dosing (e.g.,at 65 IU/kg). “Therapeutic dose,” as used herein, means a dose thatachieves a therapeutic goal, as described herein. The calculation of therequired dosage of factor VIII is based upon the empirical finding that,on average, 1 IU of factor VIII per kg body weight raises the plasmafactor VIII activity by approximately 2 IU/dL. The required dosage isdetermined using the following formula:

Required units=body weight (kg)×desired factor VIII rise (IU/dL or % ofnormal)×0.5 (IU/kg per IU/dL)

The therapeutic doses that may be used in the methods of the inventionare about 10-100 IU/kg, more specifically, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, or 90-100 IU/kg, and more specifically, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 IU/kg.

Additional therapeutic doses that may be used in the methods of theinvention are about 10 to about 150 IU/kg, more specifically, about100-110, 110-120, 120-130, 130-140, 140-150 IU/kg, and morespecifically, about 110, 115, 120, 125, 130, 135, 140, 145, or 150IU/kg.

“Variant,” as used herein, refers to a polynucleotide or polypeptidediffering from the original polynucleotide or polypeptide, but retainingessential properties thereof, e.g., factor VIII coagulant activity or Fc(FcRn binding) activity. Generally, variants are overall closelysimilar, and, in many regions, identical to the original polynucleotideor polypeptide. Variants include, e.g., polypeptide and polynucleotidefragments, deletions, insertions, and modified versions of originalpolypeptides.

Variant polynucleotides may comprise, or alternatively consist of, anucleotide sequence which is at least 85%, 90%, 95%, 96%, 97%, 98% or99% identical to, for example, the nucleotide coding sequence in SEQ IDNO:1, 3, or 5 (the factor VIII portion, the Fc portion, individually ortogether) or the complementary strand thereto, the nucleotide codingsequence of known mutant and recombinant factor VIII or Fc such as thosedisclosed in the publications and patents cited herein or thecomplementary strand thereto, a nucleotide sequence encoding thepolypeptide of SEQ ID NO:2, 4, or 6 (the factor VIII portion, the Fcportion, individually or together), and/or polynucleotide fragments ofany of these nucleic acid molecules (e.g., those fragments describedherein). Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions or lower stringency conditionsare also included as variants, as are polypeptides encoded by thesepolynucleotides as long as they are functional.

Variant polypeptides may comprise, or alternatively consist of, an aminoacid sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to, for example, the polypeptide sequence shown in SEQ IDNOS:2, 4, or 6 (the factor VIII portion, the Fc portion, individually ortogether), and/or polypeptide fragments of any of these polypeptides(e.g., those fragments described herein).

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence, it is intended thatthe nucleotide sequence of the nucleic acid is identical to thereference sequence except that the nucleotide sequence may include up tofive point mutations per each 100 nucleotides of the referencenucleotide sequence. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may be,for example, the entire sequence shown in SEQ ID NO:1 or 3, the ORF(open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toa nucleotide sequence or polypeptide of the present invention can bedetermined conventionally using known computer programs. In oneembodiment, a method for determining the best overall match between aquery sequence (reference or original sequence) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe FASTDB computer program based on the algorithm of Brutlag et al.,Comp. App. Biosci. 6:237-245 (1990), which is herein incorporated byreference in its entirety In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. In another embodiment, parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequences of SEQ ID NO:2 (the factor VIII portion, the Fcportion, individually or together) or 4, or a known factor VIII or Fcpolypeptide sequence, can be determined conventionally using knowncomputer programs. In one embodiment, a method for determining the bestoverall match between a query sequence (reference or original sequence)and a subject sequence, also referred to as a global sequence alignment,can be determined using the FASTDB computer program based on thealgorithm of Brutlag et al., Comp. App. Biosci. 6:237-245(1990),incorporated herein by reference in its entirety. In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. In another embodiment,parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0,k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization GroupLength=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5,Gap Size Penalty=0.05, Window Size=500 or the length of the subjectamino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The polynucleotide variants may contain alterations in the codingregions, non-coding regions, or both. In one embodiment, thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In another embodiment,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. In other embodiments, variants in which5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in anycombination. Polynucleotide variants can be produced for a variety ofreasons, e.g., to optimize codon expression for a particular host(change codons in the human mRNA to others, e.g., a bacterial host suchas E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides. For instance, one or more aminoacids can be deleted from the N-terminus or C-terminus of the secretedprotein without substantial loss of biological function. Ron et al., J.Biol. Chem. 268: 2984-2988 (1993), incorporated herein by reference inits entirety, reported variant KGF proteins having heparin bindingactivity even after deleting 3, 8, or 27 amino-terminal amino acidresidues. Similarly, Interferon gamma exhibited up to ten times higheractivity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988), incorporated herein by reference in its entirety.)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993),incorporated herein by reference in its entirety) conducted extensivemutational analysis of human cytokine IL-1a. They used randommutagenesis to generate over 3,500 individual IL-1a mutants thataveraged 2.5 amino acid changes per variant over the entire length ofthe molecule. Multiple mutations were examined at every possible aminoacid position. The investigators found that “[m]ost of the moleculecould be altered with little effect on either [binding or biologicalactivity].” (See Abstract.) In fact, only 23 unique amino acidsequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

As stated above, polypeptide variants include, e.g., modifiedpolypeptides. Modifications include, e.g., acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporatedherein by reference in its entirety), proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. In some embodiments, Factor VIII ismodified, e.g., pegylated, at any convenient location. In someembodiments, Factor VIII is pegylated at a surface exposed amino acid ofFactor VIII, e.g., a surface exposed cysteine, which may be anengineered cysteine. Id. In some embodiments, modified Factor VIII,e.g., pegylated Factor VIII, is a long-acting Factor VIII.

“Volume of distribution at steady state (Vss),” as used herein, has thesame meaning as the term used in pharmacology, which is the apparentspace (volume) into which a drug distributes. Vss=the amount of drug inthe body divided by the plasma concentration at steady state.

“About” as used herein for a range, modifies both ends of the range.Thus, “about 10-20” means “about 10 to about 20.”

The chimeric polypeptide used herein can comprise processed Factor VIIIor single chain Factor VIII or a combination thereof “Processed FactorVIII,” as used herein means Factor VIII that has been cleaved atArginine 1648 (for full-length Factor VIII) or Arginine 754 (forB-domain deleted Factor VIII), i.e., intracellular processing site. Dueto the cleavage at the intracellular processing site, processed FactorVIII comprises two polypeptide chains, the first chain being a heavychain and the second chain being a light chain. For example, theprocessed Factor VIII-Fc fusion protein (i.e., Heavy chain and Lightchain fused to Fc) run at approximately 90 kDa and 130 kDa on anon-reducing SDS-PAGE, respectively, and 90 kDa and 105 kDa on areducing SDS-PAGE, respectively. Therefore, in one embodiment, at leastabout 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100% of the Factor VIII portion in the chimeric polypeptide is processedFactor VIII. In another embodiment, about 50%, about 60%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100% of the Factor VIII portion inthe chimeric polypeptide is processed Factor VIII. In a particularembodiment, the chimeric polypeptide comprising processed Factor VIII ispurified (or isolated) from the chimeric polypeptide comprising singlechain Factor VIII, and at least about 90%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100% of the Factor VIII portion inthe chimeric polypeptide is processed Factor VIII.

“Single chain Factor VIII,” “SC Factor VIII,” or “SCFVIII” as usedherein means Factor VIII that has not been cleaved at the Arginine site(residue 1648 for full-length Factor VIII (i.e., residue 1667 of SEQ IDNO: 6) or residue 754 for B-domain deleted Factor VIII (i.e., residue773 of SEQ ID NO: 2). Therefore, single chain Factor VIII in thechimeric polypeptide used herein comprises a single chain. In oneembodiment, the single chain Factor VIII contains an intactintracellular processing site. In another embodiment, the single chainFactor VIII of the invention comprises a substitution or mutation at anamino acid position corresponding to Arginine 1645, a substitution ormutation at an amino acid position corresponding to Arginine 1648, or asubstitution or mutation at amino acid positions corresponding toArginine 1645 and Arginine 1648 in full-length Factor VIII. In otherembodiments, the amino acid substituted at the amino acid positioncorresponding to Arginine 1645 is a different amino acid from the aminoacid substituted at the amino acid position corresponding to Arginine1648. In certain embodiments, the substitution or mutation is an aminoacid other than arginine, e.g., isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan, valine, alanine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline,selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, ortaurine. The single chain Factor VIII-Fc fusion protein can run atapproximately 220 kDa on a non reducing SDS-PAGE and at approximately195 kDa on a reducing SDS-PAGE.

In one embodiment, the chimeric polypeptide comprising single chainFactor VIII is purified (or isolated) from the chimeric polypeptidecomprising processed Factor VIII, and at least about 30%, about 40%,about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, about 99%, or about 100% of the Factor VIII portion ofthe chimeric polypeptide used herein is single chain Factor VIII. Inanother embodiment, at least about 1%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, or about 35% of the Factor VIII portionof the chimeric polypeptide is single chain Factor VIII. In otherembodiments, about 1%-about 10%, about 5%-about 15%, about 10%-about20%, about 15%-about 25%, about 20%-about 30%, about 25%-about 35%,about 30%-about 40% of the Factor VIII portion of the chimericpolypeptide used herein is single chain Factor VIII. In a particularembodiment, about 1%, about 5%, about 10%, about 15%, about 20% about25%, about 30%, about 35% of the Factor VIII portion of the chimericpolypeptide used herein is single chain Factor VIII. In otherembodiments, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100% of the Factor VIII portion of the chimericpolypeptide used herein is single chain Factor VIII. In someembodiments, the ratio of the single chain Factor VIII to the processedFactor VIII of the chimeric polypeptide is (a) about 25% of single chainFactor VIII and about 75% of processed Factor VIII; (b) about 20% ofsingle chain Factor VIII and about 80% of processed Factor VIII; (c)about 15% of single chain Factor VIII and about 85% of processed FactorVIII; (d) about 10% of single chain Factor VIII and about 90% ofprocessed Factor VIII; (e) about 5% of single chain Factor VIII andabout 95% of processed Factor VIII; (f) about 1% of single chain FactorVIII and about 99% of processed Factor VIII; (g) about 100% of processedFactor VIII, (h) about 30% of single chain Factor VIII and about 70% ofprocessed Factor VIII, (i) about 35% of single chain Factor VIII andabout 65% of processed Factor VIII, or (j) about 40% of single chainFactor VIII and about 60% of processed Factor VIII. In otherembodiments, the ratio of the single chain Factor VIII to the processedFactor VIII of the chimeric polypeptide is (a) about 30% of single chainFactor VIII and about 70% of processed Factor VIII; (b) about 40% ofsingle chain Factor VIII and about 60% of processed Factor VIII; (c)about 50% of single chain Factor VIII and about 50% of processed FactorVIII; (d) about 60% of single chain Factor VIII and about 40% ofprocessed Factor VIII; (e) about 70% of single chain Factor VIII andabout 30% of processed Factor VIII; (f) about 80% of single chain FactorVIII and about 20% of processed Factor VIII; (g) about 90% of singlechain Factor VIII and about 10% of processed Factor VIII; (h) about 95%of single chain Factor VIII and about 5% of processed Factor VIII; (i)about 99% of single chain Factor VIII and about 1% of processed FactorVIII; or (j) about 100% of single chain Factor VIII.

The Factor VIII portion in the chimeric polypeptide used herein hasFactor VIII activity. Factor VIII activity can be measured by any knownmethods in the art. For example, one of those methods can be achromogenic assay. The chromogenic assay mechanism is based on theprinciples of the blood coagulation cascade, where activated Factor VIIIaccelerates the conversion of Factor X into Factor Xa in the presence ofactivated Factor IX, phospholipids and calcium ions. The Factor Xaactivity is assessed by hydrolysis of a p-nitroanilide (pNA) substratespecific to Factor Xa. The initial rate of release of p-nitroanilinemeasured at 405 nM is directly proportional to the Factor Xa activityand thus to the Factor VIII activity in the sample. The chromogenicassay is recommended by the Factor VIII and Factor IX Subcommittee ofthe Scientific and Standardization Committee (SSC) of the InternationalSociety on Thrombosis and Hemostatsis (ISTH). Since 1994, thechromogenic assay has also been the reference method of the EuropeanPharmacopoeia for the assignment of FVIII concentrate potency. Thus, inone embodiment, the chimeric polypeptide comprising single chain FactorVIII has Factor VIII activity comparable to a chimeric polypeptidecomprising processed Factor VIII (e.g., a chimeric polypeptideconsisting essentially of or consisting of two Fc portions and processedFactor VIII, wherein said processed Factor VIII is fused to one of thetwo Fc portions), when the Factor VIII activity is measured in vitro bya chromogenic assay.

In another embodiment, the chimeric polypeptide comprising single chainFactor VIII of this invention has a Factor Xa generation rate comparableto a chimeric polypeptide comprising processed Factor VIII (e.g., achimeric polypeptide consisting essentially of or consisting of two Fcportions and processed Factor VIII, wherein the processed Factor VIII isfused to one Fc of the two Fc portions).

In order to activate Factor X to Factor Xa, activated Factor IX (FactorIXa) hydrolyses one arginine-isoleucine bond in Factor X to form FactorXa in the presence of Ca2+, membrane phospholipids, and a Factor VIIIcofactor. Therefore, the interaction of Factor VIII with Factor IX iscritical in coagulation pathway. In certain embodiments, the chimericpolypeptide comprising single chain factor VIII can interact with FactorIXa at a rate comparable to a chimeric polypeptide comprising processedFactor VIII (e.g., a chimeric polypeptide consisting essentially of orconsisting of two Fc portions and processed Factor VIII, wherein theprocessed Factor VIII is fused to one Fc of the two Fc portions).

In addition, Factor VIII is bound to von Willebrand Factor whileinactive in circulation. Factor VIII degrades rapidly when not bound tovWF and is released from vWF by the action of thrombin. In someembodiments, the chimeric polypeptide comprising single chain FactorVIII binds to von Willebrand Factor at a level comparable to a chimericpolypeptide comprising processed Factor VIII (e.g., a chimericpolypeptide consisting essentially of or consisting of two Fc portionsand processed Factor VIII, wherein the processed Factor VIII is fused toone Fc of the two Fc portions).

Factor VIII can be inactivated by activated protein C in the presence ofcalcium and phospholipids. Activated protein C cleaves Factor VIII heavychain after Arginine 336 in the A1 domain, which disrupts a Factor Xsubstrate interaction site, and cleaves after Arginine 562 in the A2domain, which enhances the dissociation of the A2 domain as well asdisrupts an interaction site with the Factor IXa. This cleavage alsobisects the A2 domain (43 kDa) and generates A2-N (18 kDa) and A2-C (25kDa) domains. Thus, activated protein C can catalyze multiple cleavagesites in the heavy chain. In one embodiment, the chimeric polypeptidecomprising single chain Factor VIII is inactivated by activated ProteinC at a level comparable to a chimeric polypeptide comprising processedFactor VIII (e.g., a chimeric polypeptide consisting essentially of orconsisting of two Fc portions and processed Factor VIII, wherein theprocessed Factor VIII is fused to one Fc of the two Fc portions).

In other embodiments, the chimeric polypeptide comprising single chainFactor VIII has Factor VIII activity in vivo comparable to a chimericpolypeptide comprising processed Factor VIII (e.g., a chimericpolypeptide consisting essentially of or consisting of two Fc portionsand processed Factor VIII, wherein the processed Factor VIII is fused toone Fc of the two Fc portions). In a particular embodiment, the chimericpolypeptide comprising single chain Factor VIII is capable of protectinga HemA mouse at a level comparable to a chimeric polypeptide comprisingprocessed Factor VIII (e.g., a chimeric polypeptide consistingessentially of or consisting of two Fc portions and processed FactorVIII, wherein said processed Factor VIII is fused to one Fc of the twoFc portions) in a HemA mouse tail vein transection model.

The term “comparable” as used herein means a compared rate or levelresulted from using the chimeric polypeptide is equal to, substantiallyequal to, or similar to the reference rate or level. The term “similar”as used herein means a compared rate or level has a difference of nomore than 10% or no more than 15% from the reference rate or level(e.g., FXa generation rate by a chimeric polypeptide consistingessentially of or consisting of two Fc portions and processed FactorVIII, wherein said processed Factor VIII is fused to one Fc of the twoFc portions). The term “substantially equal” means a compared rate orlevel has a difference of no more than 0.01%, 0.5% or 1% from thereference rate or level.

The present invention further includes a composition comprising achimeric polypeptide having Factor VIII activity, wherein at least about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%,about 90%, about 95%, or about 99% of the chimeric polypeptide comprisesa Factor VIII portion, which is single chain Factor VIII and a secondportion. In another embodiment, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%,about 97%, about 98%, or about 99% of the chimeric polypeptide in thecomposition is single chain Factor VIII. In other embodiments, thesecond portion is an Fc, XTEN, albumin, a PAS sequence, transferrin, CTP(28 amino acid C-terminal peptide (CTP) of hCG with its 4 O-glycans),polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin bindingpolypeptide, albumin-binding small molecules, or two or morecombinations thereof. In still other embodiments, the composition of thepresent invention comprises a combination of a chimeric polypeptidecomprising processed Factor VIII and a chimeric polypeptide comprisingsingle chain Factor VIII, (a) wherein about 30% of the Factor VIIIportion of the chimeric polypeptide is single chain Factor VIII, andabout 70% of the Factor VIII portion of the chimeric polypeptide isprocessed Factor VIII; (b) wherein about 40% of the Factor VIII portionof the chimeric polypeptide is single chain Factor VIII, and about 60%of the Factor VIII portion of the chimeric polypeptide is processedFactor VIII; (c) wherein about 50% of the Factor VIII portion of thechimeric polypeptide is single chain Factor VIII, and about 50% of theFactor VIII portion of the chimeric polypeptide is processed FactorVIII; (d) wherein about 60% of the Factor VIII portion of the chimericpolypeptide is single chain Factor VIII and about 40% of the Factor VIIIportion of the chimeric polypeptide being processed Factor VIII; (e)wherein about 70% of the Factor VIII portion of the chimeric polypeptideis single chain Factor VIII and about 30% of the Factor VIII portion ofthe chimeric polypeptide is processed Factor VIII; (f) wherein about 80%of the Factor VIII portion of the chimeric polypeptide is single chainFactor VIII and about 20% of the Factor VIII portion of the chimericpolypeptide is processed Factor VIII; (g) wherein about 90% of theFactor VIII portion of the chimeric polypeptide is single chain FactorVIII and about 10% of the Factor VIII portion of the chimericpolypeptide is processed Factor VIII; (h) wherein about 95% of theFactor VIII portion of the chimeric polypeptide is single chain FactorVIII and about 5% of the Factor VIII portion of the chimeric polypeptideis processed Factor VIII; (i) wherein about 99% of the Factor VIIIportion of the chimeric polypeptide is single chain Factor VIII andabout 1% of the Factor VIII portion of the chimeric polypeptide isprocessed Factor VIII; or (j) wherein about 100% of the Factor VIIIportion of the chimeric polypeptide is single chain Factor VIII.

In certain embodiments, the composition of the present invention hasFactor VIII activity comparable to the composition comprising processedFactor VIII (e.g., a composition comprising a chimeric polypeptide,which consists essentially of or consists of two Fc portions andprocessed Factor VIII, wherein said processed Factor VIII is fused toone of the two Fc portions), when the Factor VIII activity is measuredin vitro by a chromogenic assay.

In other embodiments, the composition of the invention has a Factor Xageneration rate comparable to a composition comprising processed FactorVIII (e.g., a composition comprising a chimeric polypeptide, whichconsists essentially of or consists of two Fc portions and processedFactor VIII, wherein the processed Factor VIII is fused to one Fc of thetwo Fc portions). In still other embodiments, the composition comprisingsingle chain factor VIII can interact with Factor IXa at a ratecomparable to a composition comprising processed Factor VIII (e.g., acomposition comprising a chimeric polypeptide, which consistsessentially of or consists of two Fc portions and processed Factor VIII,wherein the processed Factor VIII is fused to one Fc). In furtherembodiments, the single chain Factor VIII in the chimeric polypeptide ofthe present composition is inactivated by activated Protein C at a levelcomparable to processed Factor VIII in a chimeric polypeptide of acomposition (e.g., a composition comprising a chimeric polypeptide,which consists essentially of or consists of two Fc portions andprocessed Factor VIII, wherein the processed Factor VIII is fused to oneFc of the two Fc portions). In a particular embodiment, the compositioncomprising single chain Factor VIII has Factor VIII activity in vivocomparable to the composition comprising processed Factor VIII (e.g., acomposition comprising a chimeric polypeptide, which consistsessentially of or consists of two Fc portions and processed Factor VIII,wherein the processed Factor VIII is fused to one Fc of the two Fcportions). In some embodiments, the composition comprising single chainFactor VIII of the invention is capable of protecting HemA mouse at alevel comparable to the composition comprising processed Factor VIII(e.g., a composition comprising a chimeric polypeptide, which consistsessentially of or consists of two Fc portions and processed Factor VIII,wherein said processed Factor VIII is fused to one Fc of the two Fcportions) in HemA mouse tail vein transection model.

The present invention further provides a method for treating a bleedingcondition in a human subject using the composition of the invention. Anexemplary method comprises administering to the subject in need thereofa therapeutically effective amount of a pharmaceuticalcomposition/formulation comprising a chimeric polypeptide having FactorVIII activity, wherein at least about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%of the chimeric polypeptide comprises a Factor VIII portion, which issingle chain Factor VIII, and a second portion.

The bleeding condition can be caused by a blood coagulation disorder. Ablood coagulation disorder can also be referred to as a coagulopathy. Inone example, the blood coagulation disorder, which can be treated with apharmaceutical composition of the current disclosure, is hemophilia orvon Willebrand disease (vWD). In another example, the blood coagulationdisorder, which can be treated with a pharmaceutical composition of thepresent disclosure is hemophilia A.

In some embodiments, the type of bleeding associated with the bleedingcondition is selected from hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath.

In other embodiments, the subject suffering from bleeding condition isin need of treatment for surgery, including, e.g., surgical prophylaxisor peri-operative management. In one example, the surgery is selectedfrom minor surgery and major surgery. Exemplary surgical proceduresinclude tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranialsurgery, intra-abdominal surgery, intrathoracic surgery, jointreplacement surgery (e.g., total knee replacement, hip replacement, andthe like), heart surgery, and caesarean section.

In another example, the subject is concomitantly treated with FIX.Because the compounds of the invention are capable of activating FIXa,they could be used to pre-activate the FIXa polypeptide beforeadministration of the FIXa to the subject.

The methods of the invention may be practiced on a subject in need ofprophylactic treatment or on-demand treatment.

The pharmaceutical compositions comprising at least 30% of single chainFactor VIII may be formulated for any appropriate manner ofadministration, including, for example, topical (e.g., transdermal orocular), oral, buccal, nasal, vaginal, rectal or parenteraladministration.

The term parenteral as used herein includes subcutaneous, intradermal,intravascular (e.g., intravenous), intramuscular, spinal, intracranial,intrathecal, intraocular, periocular, intraorbital, intrasynovial andintraperitoneal injection, as well as any similar injection or infusiontechnique The composition can be also for example a suspension,emulsion, sustained release formulation, cream, gel or powder. Thecomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides.

In one example, the pharmaceutical formulation is a liquid formulation,e.g., a buffered, isotonic, aqueous solution. In another example, thepharmaceutical composition has a pH that is physiologic, or close tophysiologic. In other examples, the aqueous formulation has aphysiologic or close to physiologic osmolarity and salinity. It cancontain sodium chloride and/or sodium acetate. In some examples, thecomposition of the present invention is lyophilized.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention. All patents and publicationsreferred to herein are expressly incorporated by reference.

EXAMPLES Example 1 Cloning, Expression and Purification of rFVIIIFc

All molecular biology procedures were performed following standardtechniques. The coding sequence of human FVIII (Genbank accession numberNM_(—)000132), including its native signal sequence, was obtained byreverse transcription-polymerase chain reactions (RT-PCR) from humanliver polyA RNA. Due to the large size of FVIII, the coding sequence wasobtained in several sections from separate RT-PCR reactions, andassembled through a series of PCR reactions, restriction digests andligations into an intermediate cloning vector containing a B domaindeleted (BDD) FVIII coding region with a fusion of serine 743 (S743) toglutamine 1638 (Q1638), eliminating 2682 bp from the B domain of fulllength FVIII. The human IgG1 Fc sequence (e.g., GenBank accession numberY14735) was obtained by PCR from a leukocyte cDNA library, and the finalexpression cassette was made in such a way that the BDD FVIII sequencewas fused directly to the N-terminus of the Fc sequence (hinge, CH2 andCH3 domains, beginning at D221 of the IgG1 sequence, EU numbering) withno intervening linker. For expression of the Fc chain alone, the mouseIgκ (kappa) light chain signal sequence was created with syntheticoligonucleotides and added to the Fc coding sequence using PCR to enablesecretion of this protein product. The FVIIIFc and Fc chain codingsequences were cloned into a dual expression vector, pBudCE4.1(Invitrogen, Carlsbad, Calif.).

HEK 293H cells (Invitrogen, Carlsbad, Calif.) were transfected with thepSYN-FVIII-013 plasmid using Lipofectamine transfection reagent(Invitrogen, Carlsbad, Calif.)), and a stable cell line was selectedwith zeocin. Cells were grown in serum free suspension culture, andrFVIIIFc protein purified from clarified harvest media using a fourcolumn purification process, including a FVIII-specific affinitypurification step (McCue J. et al., J. Chromatogr. A., 1216(45): 7824-30(2009)), followed by a combination of anion exchange columns and ahydrophobic interaction column.

Example 2 Biochemical Characterization

Processed recombinant FVIII-Fc (rFVIIIFc) is synthesized as twopolypeptide chains, one chain consisting of BDD-FVIII (S743-Q1638fusion, 1438 amino acids) fused to the Fc domain (hinge, CH2 and CH3domains) of IgG1 (226 amino acids, extending from D221 to G456, EUnumbering), for a total chain length of 1664 amino acids, the otherchain consisting of the same Fc region alone (226 amino acids). Thoughcells transfected with the FVIIIFc/Fc dual expression plasmid wereexpected to secrete three products (FVIIIFc dimer, FVIIIFc monomer, andFc dimer), only the FVIIIFc monomer and Fc dimer were detected inconditioned media. Purified FVIIIFc was analyzed by non-reducing andreducing SDS-PAGE analysis (FIGS. 2A and, B). For the nonreducedSDS-PAGE, bands were found migrating at approximately 90 kDa and 130kDa, consistent with the predicted molecular weights of the FVIIIFcheavy chain (HC) and light chain-dimeric Fc fusion (LCFc2) (FIG. 2A,lane 3). A third band was also detected at approximately 220 kDa,consistent with the predicted molecular weight for single chain FVIIIFc(SC FVIIIFc; HC+LCFc2), in which the arginine residue at position 754(1648 with respect to the full length sequence) is not cleaved duringsecretion. For the reduced SDS-PAGE analysis, major bands were seenmigrating at approximately 25 kDa, 90 kDa, 105 kDa, and 195 kDa,consistent with the predicted molecular weights for the single chain Fc,HC, LCFc, and SC FVIIIFc (FIG. 2B, lane 3). Cotransfection with humanPC5, a member of the proprotein convertase subtlisin/kexin (PCSK) typeproteases, resulted in full processing of the rFVIIIFc product (FIG. 2A,B, lane 2).

Densitometry analysis of several batches of rFVIIIFc after SDS-PAGEindicated greater than 98% purity of the expected bands. Size exclusionchromatography (SEC) was also used to assess the degree of aggregationpresent, and all batches were found to have aggregate levels at 0.5% orless.

rFVIIIFc structure was further analyzed by thrombin cleavage, reduction,and analysis by LC/UV and LC/MS. The four Factor VIII fragmentsgenerated by thrombin (by cleavages at three arginine residues, atpositions 372, 740 and 795 (795 corresponds to 1689 with respect to thefull length FVIII sequence), can be detected by UV absorbance (FIG. 2C),corresponding to the following segments of the protein: Fc (peak 1),light-chain-Fc (peak 2); the A1 domain from the heavy chain (peak 3) andthe A2 domain from the heavy chain (peak 4). The 14 amino acid B domainlinker and ˜6 kDa a3-related peptides are not detected by UV absorbancedue to their small size.

Analysis of the thrombin digestion of rFVIIIFc by HPLC/MS providedfurther detailed information of the four main domains as well as the ˜6kDa a3 related peptides, and was compared to REFACTO®, a CHO-derivedrecombinant BDD-FVIII protein (rBDD FVIII), using the same methods.FVIII samples were passed through Detergent-OUTDTG-100X columns(GBioscience, Maryland Heights, Mo.) for the removal of Tween, fullydigested with thrombin, reduced and analyzed either by RP-HPLC-UV (POROSR1/10, Applied Biosystems) or RP-HPLC-MS (Agilent 1200 coupled to anAgilent 6210TOF mass spectrometer) using gradients of acetonitrile inwater+0.1% formic acid. Peptide sequence was also confirmed with LysCpeptide mapping, analyzed by RP-HPLC/MS (Thermo Finnigan LTQ-XL-ETD).

As expected, the total ion current (TIC) chromatogram of rFVIIIFc (FIG.2D) appears similar to the UV chromatogram (FIG. 2C). Five of theexpected six products can be detected by LC/MS, including two forms ofthe a3 acidic region generated from the processed and single chainisoforms, as well as the thrombin used for the digestion. An additionaltruncated form of the a3 acidic region was also observed, and isdescribed more fully below. rBDD FVIII yielded a similar TICchromatogram, but without the free Fc chain and having a different massfor the LC compared to the rFVIIIFc LC-Fc, consistent with the lack ofan Fc region (data not shown).

Due to the heterogeneity of glycosylation over much of the protein, thedeconvoluted mass spectra for the A1, LC-Fc, and Fc regions are complexand therefore the identity of all of the molecular ions have not beenestablished. However, the observed mass for the three major peaks fromthe Fc region were found to match the G0, G1, and G2 isoforms found onIgG molecules, corresponding to biantennary oligosaccharides terminatingin 0, 1 or 2 galactose residues. The deconvoluted mass spectra of thea3-related peptides and the A2 domain provide the most definitive data,as there are no heterogeneity in the posttranslational modifications inthese regions, allowing the expected masses to be identifiedunambiguously.

The 6 kDa N-terminal peptide released from the LC after cleavage ofR1689 is predicted to comprise the a3 acidic region (amino acids E1649to R1689) if derived from the processed isoform, and the 14 amino acidtruncated B domain fused to the a3 acidic region if derived from thesingle chain isoform. Both rFVIIIFc and rBDD FVIII were found to containboth forms of the a3 region, proportional to the expected levels basedon SDS-PAGE analysis. In addition, both proteins contained a truncatedform of the a3 region corresponding to amino acids D1658-R1689, as hasbeen reported for other FVIII products, though this was found in greaterabundance in rBDD FVIII than in rFVIIIFc.

The A2 domain contains three potential tyrosine sulfation sites, but noglycosylation sites that could result in complex heterogeneity, andtherefore the exact masses of this region can be calculated. In additionto the primary expected peak in the deconvoluted mass spectrum ofrFVIIIFc correlating to the mass of the S373 to R740 sequence (FIG. 2E),two additional forms were identified corresponding to known truncationsof the FVIII HC, correlating with an A2 domain truncated at E720 andY729. These reported truncated forms were also observed directly in thedeconvoluted spectrum of rBDD FVIII A2 (FIG. 2E). Both rFVIIIFc and rBDDFVIII A2 contained similar relative amounts of the form truncated atY729 while the rBDD FVIII A2 domain contained a notably greater level ofthe form truncated at E720 as compared to the same form in rFVIIIFc(FIG. 2E).

The primary sequence of rFVIIIFc was confirmed by peptide mapping withlysyl endopeptidase (Lys-C) digests followed by UV and massspectrometric detection. Of the 99 theoretical peptides produced fromrFVIIIFc, 81 were detected, corresponding to 98% of the total sequence.The posttranslational modifications of rFVIIIFc were also characterizedby this method. FVIII contains 6 potential tyrosine sulfation sites,corresponding to positions 346, 718, 719, 723, 1664, and 1680. Fullysulfated peptides corresponding to these six sites were found, withtrace amounts of non-sulfated peptide corresponding to position 1680 asassessed by integration of the total ion chromatogram in the massspectra, and no detectable non-sulfated peptides corresponding to theother positions. BDD FVIII also contains 6 potential N-glycosylationsites, four of which have been reported to be glycosylated inrecombinant FVIII products. Consistent with this, rFVIIIFc was found tohave the same 4 sites glycosylated; N239 and N2118 were found to containhigh mannose structures, while N41 and N1810 were found to contain morecomplex carbohydrates, similar to those found on rBDD FVIII. The Fcregion N-linked glycosylation was found to match the G0, G1, and G2isoforms found with the thrombin map by LC/MS. FVIII has been reportedto have 0-glycosylation sites at Ser 741 and 743 that are partiallyoccupied, and this was found to be the case with rFVIIIFc as well.

The rFVIIIFc polypeptide produced without cotransfected processingenzymes exhibited 15-25% single chain FVIIIFc (SC FVIIIFc), whichdiffers from processed rFVIIIFc by a single peptide bond between R754and E755 (R1648/E1649 with respect to the full length FVIII). Thisisoform was purified and characterized in all of the biochemical assaysdescribed above, and found to be comparable to rFVIIIFc as shown below.The activity of purified single chain FVIIIFc was found to be similar torFVIIIFc in a chromogenic assay as well as by the various functionalassays described below.

Measurement of FVIII Activity by Chromogenic and One-Stage aPTT Assays

FVIII activity was measured by a FVIII chromogenic assay. The averagespecific activity from four separate batches of rFVIIIFc was found to be9762±449 IU/mg by the chromogenic assay, corresponding to 2148±99IU/nmol. The average specific activity from fourteen separate batches ofrFVIIIFc was found to be 8460±699 IU/mg by the aPTT assay, and 9348±1353IU/mg by the chromogenic assay, corresponding to 1861±154 and 2057±298IU/nmol, respectively. FVIII activity of single chain FVIII:Fc was alsomeasured by the chromogenic assay and compared to the completelyprocessed rFVIII:Fc or rFVIII:Fc DS (containing about 25% single chainrFVIII:Fc). As Table 3A shows, single chain rFVIIIFc showed nosignificant difference in FVIII activity compared to the Factor VIIIactivity of completely processed FVIIIFc or rFVIIIFc DS by thechromogenic assay, both in the presence and the absence of vonWillebrand Factor (VWF). Table 3B shows that full activity ofSCrFVIIIFc, as measured by one-stage activated partial thromboplastintime (aPTT) assay, was observed in the absence of VWF.

TABLE 3A FVIII Activity by Chromogenic Assay Chromogenic Specific MatrixSample Activity(IU/mg) % CV* FVIII depleted rFVIIIFcDS (25% NP)(RECD-19189-09-013) 9066 2.49 plasma Single chain rFVIIIFc (purifiedfrom RECD 8194 2.72 19189-09-013) Completely Processed rFVIIIFc 95778.34 (purified from an engineered cell line) FVIII and vWF rFVIIIFcDS(25% NP) (RECD-19189-09-013) 10801 8.92 depleted Single chain rFVIIIFc9498 4.70 plasma (purified from RECD 19189-09-013) Completely ProcessedrFVIIIFc 9569 4.54 (purified from an engineered cell line) FVIII/VWF-rFVIIIFc 9982 4.3 depleted SC rFVIIIFc 8984 4.6 plasmaCompletely-processed rFVIIIFc 8275 8.2 supplemented with human VWF *CV =coefficient of variation

TABLE 3B FVIII Activity by aPTT assay Coagulation (aPTT) SpecificActivity Matrix Sample (IU/mg) % CV FVIII- rFVIIIFcDS (25% NP) 8210 5.88depleted (RECD-19189-09-013) plasma Single chain rFVIIIFc 3108 6.57(purified from RECD 19189-09-013) Completely Processed rFVIIIFc 86833.57 (purified from an engineered cell line) FVIII and rFVIIIFcDS (25%NP) 15621 6.47 vWF (RECD-19189-09-013) depleted Single chain rFVIIIFc13572 2.41 plasma (purified from RECD 19189-09-013) Completely ProcessedrFVIIIFc 15170 10.42 (purified from an engineered cell line) FVIII/rFVIIIFc 7742 7.4 VWF- SC rFVIIIFc 3133 4.9 depletedCompletely-processed rFVIIIFc 8495 4.0 plasma supple- mented with humanVWF

In one-stage clotting assay (APTT), SC rFVIIIFc demonstrated a 60%decrease in activity when the plasma has normal VWF level, suggestingthe potential role of VWF in the activation of SC rFVIIIFc. Thisobservation was further confirmed by addition of human VWF back to theFVIII/VWF-depleted plasma (Table 3), where the coagulant activity of SCrFVIIIFc was reduced to the same level as in congenital FVIII-deficientplasma.

Activity in Xase Complex

FVIII activity was also measured in the context of the Xase complex, byincubating activated FIX and thrombin-activated REFACTO® or rFVIIIFcprotein on a phospholipid surface in the presence of calcium, andmonitoring the conversion of FX to FXa as measured by cleavage of achromogenic or fluorogenic substrate, from which FXa generation rateswere determined. This assay was then modified by varying one componentof the assay while keeping the others constant in order to examine theinteractions with each individual component.

The FXa generation rate was determined as a function of varyingphospholipid concentrations for rFVIIIFc DS, rBDD FVIII, and singlechain rFVIIIFc (FIG. 3A), using synthetic phospholipid vesicles (25%phosphotadyl serine/75% phosphotadyl choline). Both proteins were foundto have a similar activity profile, with peak activity at approximately156 μM phospholipids.

The FXa generation rate was then determined as a function of varying FXconcentrations, and Km and Vmax values calculated (FIG. 3B). Theactivity profiles for rFVIIIFc DS, rBDD FVIII, and single chain rFVIIIFcwere found to be similar, with similar Km and Vmax (Table 4). Finally,the FXa generation rate was determined as a function of varying FIXconcentrations (FIG. 3C). The activity profiles appeared similar, withsimilar Kd and Vmax (Table 4). Similar results were obtained usingplatelets as a phospholipid source (unpublished data, June 2009).

TABLE 4 FXa Generation Parameters for FVIII Proteins on PhospholipidsLipid Source Molecule Km (nM) Vmax (nM/min) 25% PS-75% PC rFVIIIFc DS55.0 ± 5.9 65.6 ± 8.6  rBDD FVIII 51.0 ± 8.7 73.5 ± 10.1 NP rFVIIIFc53.2 ± 7.5 56.0 ± 13.8

TABLE 5 FIXa Interactions with FVIII Proteins Lipid Source Molecule Km(nM) Vmax (nM/min) 25% PS-75% PC rFVIIIFc DS 2.8 ± 0.4 4.5 ± 0.3 rBDDFVIII 2.5 ± 0.3 4.0 ± 1.0 NP rFVIIIFc 2.3 ± 0.2 3.8 ± 0.4

Inactivation by APC

Once active, FVIII is inactivated by cleavage by activated protein C(APC), as well as by dissociation of the A2 domain. rFVIIIFc and rBDDFVIII were both activated by thrombin, then incubated with APC fordifferent times and activity determined in a FXa generation assay (FIG.4). In the absence of thrombin activation, little FXa generation wasdetected, and this was increased significantly with thrombin digestion.Treatment with APC for 90 min led to a significant decrease in FXageneration rates, similar to non-activated samples, and these resultswere similar for rFVIIIFc DS, rBDD FVIII, and single chain rFVIIIFc.

Affinity for vWF

FVIII interactions with von Willebrand factor (vWF) were measured byreal-time biomolecular interaction analysis (BIAcore), based on surfacePlasmon resonance (SPR) technology, to determine the kinetics of bindingof rFVIIIFc and rBDD FVIII towards vWF (Table 6). Kinetic rateparameters of Ka (on-rate) and Kd (off-rate), and the affinity KD(Kd/Ka), were determined for each FVIII interaction under identicalconditions. Both rFVIIIFc and rBDD FVIII were found to have a low nMbinding affinity (KD) for vWF, of 1.64±0.37 and 0.846±0.181 nM,respectively. The proteins had similar off-rates, with a two folddifference in on-rate resulting in a two fold difference in theaffinity.

TABLE 6 Biocore Binding Analysis of FVIII Proteins to vWF Kinetic rateparameters Off-rate/On-rate Analyte Ligand N On-rate (M−1s−1) Off-rate(s−1) KD(M) rFVIIIFc DS hvWf 5 7.92 ± 1.51 × 10⁵ 1.25 ± 1.12 × 10⁻³ 1.64± 0.37 × 10⁻⁹ NP rFVIIIFc hvWf 5 8.66 ± 1.10 × 10⁵ 1.09 ± 0.09 × 10⁻³1.28 ± 0.22 × 10⁻⁹ rBDD FVIII hvWf 5 13.7 ± 1.50 × 10⁵ 1.14 ± 0.12 ×10⁻³ 0.846 ± 0.181 × 10⁻⁹

As shown in Table 6, the affinity of rFVIII:Fc DS or single chainrFVIIIFc with vWF was found to be in the low nM range, approximately twofold greater than that of BDD FVIII alone. At physiologicalconcentrations, this would result in a slight decrease in the percentageof rFVIIIFc (processed or single chain) complexed with vWF as comparedto free FVIII, however in vivo studies have indicated that the half lifeof rFVIIIFc is significantly prolonged over full length or BDD FVIIIdespite this slightly lower affinity, and therefore this does not appearto compromise the half life of the molecule. It may be possible that thefree rFVIIIFc is more efficiently recycled through the FcRn pathway andtherefore this may contribute to a greater prolongation of half life.

Affinity for vWF and Thrombin-Mediated Release from vWF

Recombinant B-domain deleted Factor VIIIFc (rFVIIIFc) was expressed inHEK293 cells. During biosynthesis in HEK293 cells, most of the rFVIIIFcis processed by limited proteolysis to generate a FVIII heavy chain (HC)and a FVIII light chain (LC) to which the Fc moiety is attached.Spontaneous disassociation of the HC and LC in plasma, and duringstorage of FVIII drug products, is thought to contribute to a loss ofFVIII activity. The remaining portion of the biosynthesized rFVIIIFc,which is not processed, forms a single chain isoform of rFVIIIFc (SCrFVIIIFc), which may provide enhanced manufacturability and stabilitycompared to the processed rFVIIIFc.

This example describes an assay comparing the interaction of SC rFVIIIFcwith von Willebrand factor (vWF) in relation to the interaction ofrFVIIIFc with vWF. Interactions with vWF were measured by real-timebiomolecular interaction analysis (BIAcore), based on surface Plasmonresonance (SPR) technology, to determine the kinetics of binding ofrFVIIIFc and SC rFVIIIFc towards vWF (Table 11 and FIG. 16A). FVIII-freehuman plasma derived vWF was immobilized by amine coupling on thesurface of a biosensor at levels low enough to prevent mass transportlimitation. rFVIIIFc and SC rFVIIIFc were sequentially injected insingle-cycle kinetics mode at concentrations ranging from 0.13 to 5.0nM. Sensorgram data was fit to a 1:1 interaction model.

Kinetic rate parameters of Ka (on-rate) and Kd (off-rate), and theaffinity KD (Kd/Ka), were determined for each FVIII interaction underidentical conditions. Both rFVIIIFc and SC rFVIIIFc were found to have alow nM binding affinity (KD) for vWF, of 0.34±0.1 and 0.31±0.1 nM,respectively. Both isoforms also had similar on-rates and off-rates.

TABLE 11 Biocore Binding Analysis of FVIII Proteins to vWF Off-rate/Kinetic rate parameters On-rate Analyte Ligand N On-rate (M−1s−1)Off-rate (s−1) KD(M) rFVIIIFc hvWf 6 2.6 ± 0.4 × 10⁵ 8.9 ± 1.3 × 10⁻⁴3.4 ± 0.1 × 10⁻¹⁰ SC hvWf 6 2.7 ± 0.1 × 10⁵ 8.4 ± 0.4 × 10⁻⁴ 3.1 ± 0.1 ×rFVIIIFc 10⁻¹⁰

Next, thrombin-mediated release of rFVIIIFc, SC rFVIIIFc, and B-domaindeleted FVIII lacking Fc moieties (rBDD FVIII) was measured at both 25°C. and 37° C. Human vWF was immobilized by amine coupling at similarlevels on three flow cells on the biosensor surface. The remaining flowcell served as a blank for reference purposes. The rFVIIIFc and SCrFVIIIFc proteins were captured by vWF and allowed to slowlydisassociate, and the concentrations of rFVIIIFc and SC rFVIIIFc wereadjusted to obtain equimolar capture levels by the end of thedissociation phase. Human α-thrombin solutions were prepared by 2-foldserial dilution and applied at concentrations that ranged from 0.005 to20 U/mL, resulting in proteolytic release of FVIIIa species from vWF.Thrombin mediated release of activated rFVIIIFc, SC rFVIIIFc, and rBDDFVIII from vWF was monitored in real-time using an SPR-based opticalbiosensor (FIG. 16B). Following blank reference subtraction (FIG. 16C),the release rate as a function of a-thrombin concentration wasdetermined (FIG. 16D). The thrombin half maximal effective concentration(EC₅₀) for SC rFVIIIFc was 12±1 U/mL compared to 3.9±0.3 U/mL forrFVIIIFc at 25° C. and was 15±1 U/mL for SC rFVIIIFc compared to 4.8±0.2U/mL for rFVIIIFc at 37° C. (FIG. 16E). rFVIIIFc had a similar thrombinEC₅₀ value compared to rBDD FVIII, having values of 3.9±0.3 U/mL and3.3±0.3 U/mL, respectively at 25° C. and values of 4.8±0.2 U/mL and4.0±0.2 U/mL, respectively at 37° C. SC rFVIIIFc had a thrombin EC₅₀value that was approximately 3-fold higher than rFVIIIFc. Thisimpairment of thrombin mediated release from vWF may underlie thespecific reduction in specific activity for SC rFVIIIFc observed in theaPTT assay in which vWF was present (Table 3B).

Example 3

A Phase I/IIa, open-label, crossover, dose-escalation, multi-center, andfirst-in-human study was designed to evaluate the safety, tolerability,and pharmacokinetics of a single dose of rFVIIIFc in subjects withsevere (defined as <1 IU/dL [1%] endogenous factor VIII [FVIII])hemophilia A. A total of approximately 12 previously treated patientswere enrolled and dosed with rFVIIIFc at 25 or 65 IU/kg. After thescreening (scheduled within 28 days prior to the first dose of theADVATE® [rFVIII], the reference comparator agent) and a minimum of4-days (96 hours) elapsing with no FVIII treatment prior to the firstinjection, approximately 6 subjects received a single 25 IU/kg dose ofADVATE® followed by a 3-day (72 hours) pharmacokinetic (PK) profile thencrossover and receive a 25 IU/kg single, open-label dose of rFVIIIFc fora 7-day (168 hours) PK profiling. The first 3 subjects were dosedsequentially. For the first three (3) subjects dosed with 25 IU/kg ofrFVIIIFc, each subject underwent an inhibitor assessment at 14-days (336hours) post-injection of rFVIIIFc. Dosing of the next subject (for thefirst three subjects only) occurred once the inhibitor testing iscompleted. After the 3rd subject completed the 14 day inhibitorassessment, the remaining three subjects at 25 IU/kg and the sixsubjects at 65 IU/kg began enrollment sequentially at least 1 day apartwithin each dose group.

One week after the last subject received the 25 IU/kg dose of therFVIIIFc, approximately 6 unique subjects were recruited for the 65IU/kg cohort. Each subject in the 65 IU/kg cohort received a single 65IU/kg dose of ADVATE® followed by a 4-day (96 hours) PK profiling thencrossover and receive a 65 IU/kg single, open-label dose of rFVIIIFc fora 10-day (240 hours) profiling. If a bleeding episode occurred beforethe first injection of rFVIIIFc in any cohort, subject's pre-study FVIIIproduct was used for treatment and an interval of at least 4 days had topass before receiving the first injection of rFVIIIFc for the PKprofile.

All subjects were followed for a 14-day (336 hours) and 28 day safetyevaluation period after administration of rFVIIIFc 25 IU/kg or 65 IU/kgfor safety. All subjects underwent pharmacokinetic sampling pre- andpost-dosing along with blood samples for analysis of FVIII activity atdesignated time points.

The pharmacokinetic data for the Phase I/IIa clinical trial demonstratedthe following results for FVIIIFc. FVIIIFc had about a 50% increase insystemic exposure (AUC_(INF)), about 50% reduction in clearance (Cl),and about 50-70% increase in elimination half-life and MRT compared toADVATE® (full length rFVIII). In addition, FVIIIFc showed increasedC168, TBLP1, TBLP3, and TBLP5 values compared to ADVATE®.

AUC_(INF) Area under the concentration-time curve from zero to infinityBeta HL Elimination phase half-life; also referred to as t_(1/2β) C168Estimated FVIIIFc activity above baseline at approximately 168 h afterdose Cl Clearance MRT Mean residence time TBLP1 Model-predicted timeafter dose when FVIIIFc activity has declined to approximately 1 IU/dLabove baseline TBLP3 Model-predicted time after dose when FVIIIFcactivity has declined to approximately 3 IU/dL above baseline TBLP5Model-predicted time after dose when FVIIIFc activity has declined toapproximately 5 IU/dL above baseline

Example 4

A recombinant B-domain-deleted factor VIII-Fc (rFVIIIFc) fusion proteinhas been created as an approach to extend the half-life of FVIII. Thepharmacokinetics (PK) of rFVIIIFc were compared to rFVIII in hemophiliaA mice. We found that the terminal half-life was twice as long forrFVIIIFc compared to rFVIII. In order to confirm that the underlyingmechanism for the extension of half-life was due to the protection ofrFVIIIFc by FcRn, the PK were evaluated in FcRn knockout and human FcRntransgenic mice. A single intravenous dose (125 IU/kg) was administeredand the plasma concentration measured using a chromogenic activityassay. The Cmax was similar between rFVIIIFc and rFVIII (XYNTHA®) inboth mouse strains. However, while the half-life for rFVIIIFc wascomparable to that of rFVIII in the FcRn knockout mice, the half-lifefor rFVIIIFc was extended to approximately twice longer than that forrFVIII in the hFcRn transgenic mice. These results confirm that FcRnmediates or is responsible for the prolonged half-life of rFVIIIFccompared to rFVIII. Since hemostasis in whole blood measured by rotationthromboelastometry (ROTEM®) has been shown to correlate with theefficacy of coagulation factors in bleeding models of hemophilia mice aswell as in clinical applications, we sought to evaluate the ex vivoefficacy of rFVIIIFc in the hemophilia A mice using ROTEM®. Hemophilia Amice were administered a single intravenous dose of 50 IU/kg rFVIIIFc,XYNTHA® (FVIII) or ADVATE® (FVIII). At 5 minutes post dose, clotformation was similar with respect to clotting time (CT), clot formationtime (CFT) and a-angle. However, rFVIIIFc showed significantly improvedCT at 72 and 96 hr post dose, and CFT and a-angle were also improved at96 hrs compared to both XYNTHA® (FVIII) and ADVATE® (FVIII), consistentwith prolonged PK of rFVIIIFc. Therefore construction of an Fc fusion ofFVIII produces a molecule with a defined mechanism of action that has anincreased half-life and the potential to provide prolonged protectionfrom bleeding.

Example 5

This Example presents final analysis results for FVIII activity from 16patients treated with 25 and 65 IU/kg FVIII products. See Example 3.

In this Example, rFVIIIFc is a recombinant fusion protein comprised of asingle molecule of recombinant B-domain deleted human FVIII (BDD-rFVIII)fused to the dimeric Fc domain of the human IgG1, with no interveninglinker sequence. This protein construct is also referred to herein asrFVIIIFc heterodimeric hybrid protein, FVIIIFc monomeric Fc fusionprotein, FVIIIFc monomer hybrid, monomeric FVIIIIFc hybrid, and FVIIIFcmonomer-dimer. See Example 1, FIG. 1, and Table 2A.

Preclinical studies with rFVIIIFc have shown an approximately 2-foldprolongation of the half-life of rFVIII activity compared tocommercially available rFVIII products. The rationale for this study wasto evaluate the safety and tolerability of a single dose of rFVIIIFc infrozen liquid formulation and provide data on the PK in severehemophilia A subjects. For this study, 16 evaluable subjects wereavailable for PK evaluation. Single administration of two doses of bothrFVIIIFc and ADVATE® at a nominal dose of 25 (n=6) and 65 IU/kg of bodyweight (n=10) were infused intravenously over approximately 10 minutes.Blood samples for plasma PK assessments were obtained before infusion,as well as up to 10 days after dosing. The PK of FVIII activity for bothADVATE® and rFVIIIFc were characterized in this study using amodel-dependent method.

Objectives

The primary objective of this study was to assess the safety andtolerability of single administration of two doses of rFVIIIFc (25 and65 IU/kg) in previously treated patients (PTPs) aged 12 and above withsevere hemophilia A.

The secondary objectives were to determine the pharmacokinetics (PK)parameters determined by pharmacodynamic (PD) activity of FVIII overtime after a single administration of 25 or 65 IU/kg of rFVIIIFccompared to ADVATE® in one-stage clotting and chromogenic assays.

Study Design (See Example 3)

Blood samples were collected for FVIII activity PK evaluations at thescreening visit (within 28 days prior to dosing ADVATE®); on Day 0(injection of ADVATE®) pre-injection and at 10 and 30 minutes and 1, 3,6, and 9 hours post-injection; on Day 1 at 24 hours post-injection ofADVATE®; on Day 2 at 48 hours post-injection of ADVATE®; on Day 3 at 72hours post-injection of ADVATE®; and on Day 4 at 96 hours post-injectionof high dose of ADVATE® (Cohort B only).

Blood samples were collected for FVIII activity PK evaluations on theday of rFVIIIFc injection just prior to the administration of rFVIIIFc,at 10 and 30 minutes and 1, 3, 6, and 9 hours post-injection ofrFVIIIFc; on Day 1 at 24 hours post-injection of rFVIIIFc; on Days 2through 5 at 48, 72, 96, and 120 hours post-injection of rFVIIIFc; onDay 7 at 168 hours post-injection of rFVIIIFc; on Days 8, 9, and 10 at192, 216, and 240 hours post-injection of high dose of rFVIIIFc (CohortB only). FVIII activity was also measured at the final study visit (28days post-injection of rFVIIIFc) at 672 hours post-injection ofrFVIIIFc.

Pharmacokinetic Modeling and Calculations Abbreviations

-   -   TBLP1=Model-predicted time after dose when FVIII activity has        declined to approximately 1 IU/dL above baseline.    -   TBLP3=Model-predicted time after dose when FVIII activity has        declined to approximately 3 IU/dL above baseline    -   KV_M=Cmax_M/Actual Dose (IU/kg)    -   KV_OB=Cmax_OB/Actual Dose (IU/kg)    -   IVR_M=100×Cmax_M×Plasma Volume (dL)/Total Dose in IU; where        plasma volume in mL=(23.7×Ht in cm)+(9.0×Wt in kg)−1709.    -   IVR_OB=100×Cmax_OB×Plasma Volume (dL)/Total Dose in IU; where        plasma volume in mL=(23.7×Ht in cm)+(9.0×Wt in kg)−1709.

Results Single-Dose Pharmacokinetics (One-Stage Assay)

Observed FVIII activity increased sharply after the short IV infusion ofeither ADVATE® or rFVIIIFc, with mean (±SD) model-predicted Cmax valuesof 56.6±4.74 and 121±28.2 IU/dL for ADVATE® and 55.6±8.18 and 108±16.9IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.All ADVATE®- and rFVIIIFc-treated patients had dose-related increases inFVIII activity. The observed increase in both Cmax and AUC_(INF) wasslightly less than proportional to dose over the dose range evaluated.

After the end of the infusion, the decline of the observed FVIIIactivity exhibited monoexponential decay characteristics until thebaseline level was reached. The rate of decline in FVIII activity wasslower for rFVIIIFc than for ADVATE® with mean (±SD) model-predictedelimination half-life values of 11.9±2.98 and 10.4±3.03 hr for ADVATE®and 18.0±3.88 and 18.4±6.99 hr for rFVIIIFc for the 25 and 65 IU/kg dosegroups, respectively. Elimination half-life values appeared to bedose-independent over the dose range evaluated for both FVIII products.

Total systemic FVIII exposure (assessed by AUC_(INF)) was ˜48% and 61%greater following rFVIIIFc administration than ADVATE® at 25 and 65IU/kg dose levels, respectively. Mean (±SD) model-predicted AUC_(INF)values were 974±259 and 1810±606 hr*IU/dL for ADVATE® and 1440±316 and2910±1320 hr*IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively.

Similar to elimination half-life, the MRT was prolonged for rFVIIIFcrelative to ADVATE®. Mean (±SD) model-predicted MRT values were17.1±4.29 and 14.9±4.38 hr for ADVATE® and 25.9±5.60 and 26.5±10.1 hrfor rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. MRTvalues appeared to be dose-independent over the dose range evaluated forboth FVIII products.

In addition, primary PK parameter values for CL and V were determined CLvalues for rFVIIIFc only accounted for ˜66% of those observed forADVATE® at equivalent doses. Mean (±SD) model-predicted CL values were2.70±0.729 and 4.08±1.69 mL/hr/kg for ADVATE® and 1.80±0.409 and2.69±1.25 mL/hr/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively. V values were comparable between ADVATE® and rFVIIIFc withmean (±SD) model-predicted V values of 43.9±4.27 and 56.1±13.4 mL/kg forADVATE® and 45.3±7.23 and 61.6±10.6 mL/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively. Slight increases in mean CL and Vvalues were noted with increasing dose of ADVATE® and rFVIIIFc; however,the increase in standard deviations at the 65 IU/kg dose coupled withlimited dose levels confounded an assessment of the dose-dependency ofthese parameters. For example, the CV % geometric mean CL value for therFVIIIFc treatment group increased from 23.0% (25 IU/kg) to 48.6% (65IU/kg).

In addition to the primary PK parameters, secondary PK parameters (e.g.K-values, IVR, etc.) were determined to evaluate FVIII duration ofeffect. Evidence of PK difference was also observed with rFVIIIFcdemonstrating increased TBLP1 and TBLP3 values compared to ADVATE® atequivalent doses. IVR and K-values for ADVATE® and rFVIIIFc appeared tobe comparable. A slight increase in TBLP1 and TBLP3 values were observedwith increasing dose of ADVATE® and rFVIIIFc. In contrast, slightdecreases in mean IVR and K-values were noted with increasing dose ofADVATE® and rFVIIIFc. As previously indicated, an assessment of the dosedependency of these parameters is confounded by limited dose levels.

Mean (±SD) observed TBLP1 were 2.88±0.733 and 2.93±0.848 IU/dL per IU/kgfor ADVATE® and 4.28±0.873 and 5.16±2.02 IU/dL per IU/kg for rFVIIIFcfor the 25 and 65 IU/kg dose groups, respectively. Mean (±SD) observedTBLP3 were 2.06±0.527 and 2.26±0.666 IU/dL per IU/kg for ADVATE® and3.09±0.623 and 3.93±1.59 IU/dL per IU/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively.

Mean IVR and K-values calculated using observed Cmax values (subtractedwith baseline and residual drug within the model) were generally greaterthan values determined using model-predicted Cmax values; consistentwith slight underestimation of the observed peak activity using theone-compartment model. Mean (±SD) observed K-values were 2.57±0.198 and2.13±0.598 IU/dL per IU/kg for ADVATE® and 2.46±0.330 and 1.85±0.332IU/dL per IU/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively. Mean (±SD) observed IVR values were 94.1±15.6 and85.8±16.5% for ADVATE® and 89.5±11.9 and 74.8±6.72% for rFVIIIFc for the25 and 65 IU/kg dose groups, respectively.

Single-Dose Pharmacokinetics (Chromogenic Assay)

Observed FVIII activity increased sharply after the short IV infusion ofeither ADVATE® or rFVIIIFc, with mean (±SD) model-predicted Cmax valuesof 70.2±9.60 and 157±38.6 IU/dL for ADVATE® and 70.3±10.0 and 158±34.7IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively.

All ADVATE®—and rFVIIIFc-treated patients had dose-related increases inFVIII activity. The observed increase in both Cmax and AUC_(INF) wasslightly less than proportional to dose over the dose range evaluated.

After the end of the infusion, the decline of the observed FVIIIactivity exhibited monoexponential decay characteristics until thebaseline level was reached. The rate of decline in FVIII activity wasslower for rFVIIIFc than for ADVATE® with mean (±SD) model-predictedelimination half-life values of 10.7±1.98 and 10.3±3.27 hr for ADVATE®and 16.2±2.92 and 19.0±7.94 hr for rFVIIIFc for the 25 and 65 IU/kg dosegroups, respectively. Elimination half-life values appeared to bedose-independent over the dose range evaluated for both FVIII products.

Total systemic FVIII exposure (assessed by AUC_(INF)) was ˜53% and 84%greater following rFVIIIFc administration than ADVATE® at 25 and 65IU/kg dose levels, respectively. Mean (±SD) model-predicted AUC_(INF)values were 1080±236 and 2320±784 hr*IU/dL for ADVATE® and 1650±408 and4280±1860 hr*IU/dL for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively.

Similar to elimination half-life, the MRT was prolonged for rFVIIIFcrelative to ADVATE®. Mean (±SD) model-predicted MRT values were15.3±2.86 and 14.8±4.72 hr for ADVATE® and 23.4±4.22 and 27.3±11.4 hrfor rFVIIIFc for the 25 and 65 IU/kg dose groups, respectively. MRTvalues appeared to be dose-independent over the dose range evaluated forboth FVIII products.

In addition, primary PK parameter values for CL and V were determined CLvalues for rFVIIIFc only accounted for ˜58-66% of those observed forADVATE® at equivalent doses. Mean (±SD) model-predicted CL values were2.39±0.527 and 3.21±1.40 mL/hr/kg for ADVATE® and 1.57±0.349 and1.86±0.970 mL/hr/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively. V values were comparable between ADVATE® and rFVIIIFc withmean (±SD) model-predicted V values of 35.8±5.52 and 43.6±11.2 mL/kg forADVATE® and 35.9±6.65 and 42.7±8.91 mL/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively. Increases in mean CL and V values werenoted with increasing dose of ADVATE® and rFVIIIFc; however, theincrease in standard deviations at 65 IU/kg coupled with limited doselevels confounded an assessment of the dose-dependency of theseparameters.

In addition to the primary PK parameters, secondary PK parameters (e.g.K-values, IVR, etc.) were determined to evaluate FVIII duration ofeffect. Evidence of PK difference was also observed with rFVIIIFcdemonstrating increased TBLP1 and TBLP3 values compared to ADVATE® atequivalent doses. IVR and K-values for ADVATE® and rFVIIIFc appeared tobe comparable.

A slight increase in TBLP1 and TBLP3 values were observed withincreasing dose of ADVATE® and rFVIIIFc. In contrast, slight decreasesin mean IVR and K-values were noted with increasing dose of ADVATE® andrFVIIIFc. As previously indicated, an assessment of the dose dependencyof these parameters is confounded by limited dose levels.

Mean (±SD) observed TBLP1 were 2.70±0.511 and 3.09±0.978 IU/dL per IU/kgfor ADVATE® and 4.06±0.798 and 5.66±2.38 IU/dL per IU/kg for rFVIIIFcfor the 25 and 65 IU/kg dose groups, respectively. Mean (±SD) observedTBLP3 were 1.98±0.377 and 2.39±0.718 IU/dL per IU/kg for ADVATE® and3.04±0.598 and 4.44±1.84 IU/dL per IU/kg for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively.

Mean IVR and K-values calculated using observed Cmax values (subtractedwith baseline and residual drug within the model) were generally greaterthan values determined using model-predicted Cmax values; consistentwith slight underestimation of the observed peak activity using theone-compartment model. Mean (±SD) observed K-values were 3.08±0.429 and2.85±0.721 IU/dL per IU/kg for ADVATE® and 3.12±0.451 and 2.92±0.985IU/dL per IU/kg for rFVIIIFc for the 25 and 65 IU/kg dose groups,respectively. Mean (±SD) observed IVR values were 112±14.5 and 116±26.9%for ADVATE® and 113±16.3 and 117±33.6% for rFVIIIFc for the 25 and 65IU/kg dose groups, respectively.

CONCLUSIONS

All ADVATE®—and rFVIIIFc-treated patients had comparable dose-relatedincreases in Cmax and AUCINF over the dose range evaluated. Peak plasmalevels of ADVATE® and rFVIIIFc activity were generally observed withinthe first hour after the end of the infusion and remained detectable forseveral days after dosing. After the end of infusion, the decline inbaseline corrected FVIII activity exhibited monoexponential decay untilthe baseline was reached for both products. Parameter values forelimination half-life and MRT appeared to be dose-independent over thedose range evaluated for both FVIII products. Slight increases in meanCL and V values were noted with increasing dose of ADVATE® and rFVIIIFc;however, increased intersubject variability at the 65 IU/kg coupled withlimited dose levels confounded an assessment of the dose-dependency ofthese parameters.

Comparison of rFVIIIFc and ADVATE® activity PK revealed an approximate48-61% (One-Stage Assay) or 53-84% (Chromogenic Assay) increase insystemic exposure, approximate 30-40% reduction in clearance, and anapproximate 50-80% increase in both elimination half-life and MRT forrFVIIIFc relative to ADVATE® at comparable doses. Evidence of PKdifference was also observed with rFVIIIFc demonstrating increased TBLP1and TBLP3 values compared to ADVATE® at equivalent doses. IVR andK-values for ADVATE® and rFVIIIFc appeared to be comparable.

The PK parameters obtained from the Chromogenic Assay results generallyagreed with those from the One-Stage Assay, except that the ChomogenicAssay yielded a higher estimation of exposure parameters (e.g., Cmax,AUCINF, etc.).

With the observed improvements in PK, rFVIIIFc may provide a prolongedprotection from bleeding, allowing less frequent injections forindividuals with Hemophilia A.

Example 6

On the basis of the interim PK analysis from the first in-human study ofrFVIII:Fc (Example 3), the A-LONG study was designed. A-LONG is an openlabel, multi-center evaluation of the safety, pharmacokinetics, andefficacy of recombinant Factor VIII Fc fusion (FVIII:Fc) in theprevention and treatment of bleeding in previously treated subjects withsevere hemophilia A (defined as <1 IU/dL [<1%] endogenous FVIII).

Approximately 106 subjects will be enrolled into one of three regimens:a tailored prophylaxis regimen (arm 1), a weekly dosing regimen (arm 2),and an on-demand regimen (arm 3).

Arm 1: Tailored Prophylaxis Regimen

Arm 1 will include an overall group and a PK subgroup. Approximately 66subjects will be enrolled. The initial regimen will be twice weekly at25 IU/kg on the first day, followed by 50 IU/kg on the fourth day of theweek. Subjects will administer rFVIIIFc on this weekly prophylaxisregimen until PK results for rFVIIIFc are available. Based on theseresults, a tailored prophylaxis regimen will be established for eachindividual, in which the dose and interval will be determined tomaintain a trough level of 1-3% FVIII activity. Each subject will thenadminister his individually tailored prophylaxis regimen throughout thestudy.

Subjects will be monitored throughout the study and ongoing dose andinterval adjustments will be made. Adjustments will only be made when asubject experiences unacceptable bleeding episodes defined as ≧2spontaneous bleeding episodes over a rolling two-month period. In thiscase, adjustment will target trough levels of 3-5%.

Arm 2: Weekly Dosing Regimen

Approximately 20 subjects will be enrolled/randomized and undergoabbreviated rFVIIIFc PK profiling as follows: Washout of at least 96hours; a single dose of rFVIIIFc 65 IU/kg; Abbreviated samplingbeginning on rFVIIIFc Day 0, including pre-injection and 10 (±2)minutes, 3 hours (±15 minutes), 72 (±2) hours [Day 3], and 96 (±2) hours[Day 4] from the start of injection. Following the abbreviated PKprofiling, subjects will then administer a fixed dose of 65 IU/kgrFVIIIFc every 7 days at least for 28 weeks and up to 52 weeks.

Arm 3: On-Demand Regimen

A minimum of 10 major surgeries in at least 5 subjects will be evaluatedin the study. Major surgery is defined as any surgical procedure(elective or emergent) that involves general anesthesia and/orrespiratory assistance in which a major body cavity is penetrated andexposed, or for which a substantial impairment of physical orphysiological functions is produced (e.g., laparotomy, thoracotomy,craniotomy, joint replacement, and limb amputation).

For prophylaxis during surgery, subjects will be treated with 20 to 50IU/kg rFVIIIFc every 12 to 24 hours. Prior to surgery, the physicianwill review the subject's rFVIIIFc PK profile and assess the doseregimen of Factor VIII replacement generally required for the type ofplanned surgery and the clinical status of the subject. Recommendationfor the appropriate dosing of rFVIIIFc in the surgical treatment period,including any rehabilitation time, will take these factors intoconsideration.

The primary objectives of this study are (a) to evaluate the safety andtolerability of rFVIIIFc administered as prophylaxis, on-demand, andsurgical treatment regimens; and (b) to evaluate the efficacy ofrFVIIIFc administered as prophylaxis, on-demand, and surgical treatmentregimens. The secondary objectives of this study are (a) to characterizethe PK profile of rFVIIIFc and compare the PK of FVIIIFc with thecurrently marketed product, ADVATE®; (b) to evaluate individualresponses with FVIIIFc; and (c) to evaluate FVIIIFc consumption.

Primary Objectives

-   -   To evaluate safety and tolerability of rFVIIIFc administered as        prophylaxis, weekly, on-demand, and surgical treatment regimens    -   To evaluate the efficacy of rFVIIIFc administered as tailored        prophylaxis, on-demand, and surgical treatment regimens

Secondary Objectives

-   -   To characterize the PK profile of rFVIIIFc and compare the PK of        rFVIIIFc with the currently marketed product, ADVATE®    -   To evaluate individual responses with rFVIIIFc    -   To characterize the range of dose and schedules required to        adequately prevent bleeding in a prophylaxis regimen; maintain        homeostasis in a surgical setting; or to treat bleeding episodes        in an on-demand, weekly treatment, or prophylaxis setting    -   To evaluate rFVIIIFc consumption (e.g., total annualized        rFVIIIFc consumption per subject)

Example 7 Clinical ROTEM® Assessment

In the study in Example 7, in addition to the measurement of plasmaFVIII activity by one-stage activated partial thromboplastin time (aPTT)assay, whole blood rotational thromboelastometry (ROTEM®) has also beenexplored to assess the improvement in global hemostasis by rFVIIIFc andADVATE® in 2 subjects, specifically, 1 in the low dose cohort and 1 inthe high dose cohort.

rFVIIIFc and ADVATE® appear to be comparably active in clot formationwhen spiked into subjects' blood prior to rFVIIIFc treatment. Theclotting time (CT) was linear with respect to the dose of rFVIIIFc andADVATE® in the range of approximately 1% of 100% of normal, and the doseresponse was comparable between rFVIIIFc and ADVATE® in the samesubject.

Following dosing with ADVATE® and subsequently rFVIIIFc, citrated wholeblood was sampled at various time points and the clot formationfollowing recalcification was monitored by ROTEM®. Despite the variablebaseline CT due to residue FVIII levels prior to ADVATE® or rFVIIIFcdosing, both products effectively corrected the CT to comparable levels30 minutes post-injection. In addition, the improvement in CT was bettersustained at and after 3 hours post-injection of 25 IU/kg of rFVIIIFcrelative to ADVATE® in the subject dosed at this low dose. However, thedifferential improvement of rFVIIIFc versus ADVATE® was much lessappreciable at the 65 IU/kg dose.

Example 8

HemA mice were used for tail clip studies. The mice were firstanesthetized and then injected with 4.6 μg/kg, 1.38 μg/kg, or 0.46 μg/kgof either processed rFVIIIFc (Drug Substance, which contain about75%-85% processed rFVIIIFc) and purified single chain rFVIIIFc. Afterthe injection, the tail was cut from the tip and immediately placed intoa tube to collect blood. Percentage of protection on survival wasmeasured for rFVIIIFc processed (drug substance) and single chainFVIIIFc as shown in Table 7 and FIG. 7.

TABLE 7 In Vivo Efficacy of rFVIII: Fc DS and Single chain rFVIIIFc Dose(μg/kg) 4.6 1.38 0.46 % of Protection FVIIIFc DS 93 52 19 on SurvivalSingle chain 93 64 14 rFVIIIFc

As shown in Table 7 and FIG. 7, the protection on survival by singlechain rFVIIIFc is comparable to processed rFVIIIFc (DS).

Clotting Activity by In Vitro ROTEM

The clotting potency of rFVIIIFc was further explored in whole bloodRotational

Thromboelastometry (ROTEM) over a range of concentrations, and comparedto both rBDD FVIII (Xyntha) and recombinant full length FVIII (rflFVIII;Advate). For in vitro ROTEM, rFVIII proteins were spiked in triplicateinto citrated pooled blood collected from the vena cava of 5-6 male HemAmice to the final concentration of 0, 0.1, 1, 10, and 100% of normalplasma FVIII level. The clot was initiated by the addition of CaCl₂(NATEM) and clotting time (CT), clot formation time (CFT), alpha-angleand maximum clot firmness (MCF) were recorded on the ROTEM system(Pentapharm GmbH, Munich, Germany). The clotting time (CT), clotformation time (CFT), and alpha angle for the three proteins spiked inHemA mouse blood at escalating doses from 0.1-100% of normal FVIIIlevels are shown in FIG. 14. In the wide range of 0.1 to 100% of normal,the CT and CFT are comparable among rFVIIIFc, rBDD FVIII and rflFVIII.The alpha angle is only significantly different (p<0.05) betweenrFVIIIFc and rBDD FVIII at 10%.

Clotting Activity by Ex Vivo ROTEM

The pharmacodynamics of rFVIIIFc, as measured by ROTEM, was compared torBDD FVIII and rflFVIII after a single intravenous injection intoHemophilia A mice. For ex vivo ROTEM, male HemA mice were injectedintravenously with a single dose of 50 IU/kg rFVIIIFc, ADVATE®, orXYNTHA®, and 5 mice sacrificed at each time point (5 minutes, 24, 48, 72and 96 hours post dosing). Individual citrated whole blood collectedfrom the vena cava was immediately analyzed by NATEM on the ROTEM systemand parameters measured as above. The CT, CFT, and alpha angle weredetermined for samples taken from 5 min to 96 hours after dosing, andshown in FIG. 15. At 5 min, all are comparably effective resulting insimilar CT, CFT and alpha angle (FIG. 15A-C). However, rFVIIIFcdemonstrated a significantly improved (p<0.05) CT at 72 and 96 hrs, CFTand alpha angle at 96 hrs (FIGS. 15A-C) relative to rBDD FVIII andrflFVIII.

Example 9

Recombinant factor VIIIFc (rFVIIIFc) is comprised of a B domain deleted(BDD) rFVIII protein genetically fused to the Fc domain of humanimmunoglobulin G1 (IgG1). Prior to secretion from HEK 293 cells, most ofthe rFVIIIFc is processed into a FVIII heavy chain (HC) and light chain(LC+Fc). In circulation, rFVIIIFc is complexed with von Willebrandfactor (VWF) and released upon activation in a manner that isindistinguishable from native FVIII. Spontaneous dissociation of the HCand LC is thought to contribute to the loss of FVIII activity in plasmaand during storage of FVIII drug products. Here we describe a singlechain non-processed isoform of rFVIIIFc (SC rFVIIIFc), which may providesuperior manufacturability and enhanced stability compared to nativeFVIII.

SC rFVIIIFc was purified from rFVIIIFc, which contains a fraction of thenon-processed isoform. Compared to rFVIIIFc, SC rFVIIIFc showedequivalent chromogenic activity but approximately 60% reduced activityby the one stage (aPTT) assay, (Table 3A-B). Thrombin generation assay(TGA) was performed using calibrated automated thrombogram (fromThrombinoscope®). In a thrombin generation assay (TGA), SC rFVIIIFc alsoshowed a reduced thrombin potential (FIG. 13A), and peak thrombin (FIG.13B) compared to rFVIIIFc. However, as shown in Table 3B, full activityof SC rFVIIIFc by aPTT was observed in the absence of vWF, suggestingrelease from vWF may be delayed due to covalent linkage of the a3 acidicregion to the HC after Arg 1680 cleavage in SC rFVIIIFc, in contrast toa3 release and dissociation from fully processed FVIII. Delayeddissociation from vWF may explain the reduced activity observed in theaPTT assay and TGA, while full activity was observed in the two-stagechromogenic assay. A reduced rate of activation in the presence of vWFwas confirmed in a modified chromogenic substrate assay with limitingthrombin as FVIII activator.

In vivo function of SC rFVIIIFc was assessed in the HemA mouse tail veintransection (TVT) model. HemA male mice were treated with indicateddoses of either rFVIIIFc drug product or SC rFVIIIFc 48 hours prior toTVT. Tail re-bleeding and survival were monitored hourly up to 12 hourspost TVT with final observation performed at 24-hour post TVT. SCrFVIIIFc and the rFVIIIFc demonstrated equivalent in vivo efficacy inthis model, with an ED50 of 1.17 μg/kg and 1.23 μg/kg respectively whenTVT was performed at 48 hours post infusion (FIG. 7(A)). Comparable 24hour post TVT survival curves (p≧0.65) (FIG. 7(B)) and re-bleed rates(FIG. 7(C)) in HemA mice were observed for the SC rFVIIIFc and rFVIIIFcat each tested dose level, indicating that SC rFVIIIFc was equallyeffective as rFVIIIFc despite its lower apparent aPTT activity. Thedelayed in vitro activation of SC rFVIIIFc in the presence of vWFtherefore appears to have no significant impact on its in vivo efficacy.Thus, SC rFVIIIFc represents a novel and efficacious isoform of rFVIIIFcwith potential clinical applications. Further studies will be requiredto demonstrate enhanced product stability in the context of this Fcfusion protein.

Example 10

Current factor VIII (FVIII) products display a half-life (t_(1/2)) ofapproximately 8-12 hours, requiring frequent intravenous injections forprophylaxis and treatment of hemophilia A patients. rFVIIIFc is arecombinant fusion protein composed of a single molecule of FVIIIcovalently linked to the Fc domain of human IgG₁ to extend circulatingrFVIII half-life. This first-in-human study in previously-treated malesubjects with severe hemophilia A investigated safety andpharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose ofADVATE® at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Mostadverse events were unrelated to study drug. None of the study subjectsdeveloped anti-FVIIIFc antibodies or inhibitors. Across dose levels, ascompared with ADVATE®, rFVIIIFc showed 1.54 to 1.71-fold longerelimination T_(1/2) and mean residence time, 1.49 to 1.56-fold lowerclearance, and 1.48 to 1.56-fold higher total systemic exposure. ADVATE®and rFVIIIFc had comparable dose-dependent peak plasma concentrationsand recoveries. Time to 1% FVIII activity above baseline wasapproximately 1.53 to 1.68-fold longer than ADVATE® across dose levels.Thus, rFVIIIFc may offer a viable therapeutic approach to achieveprolonged hemostatic protection and less frequent dosing in patientswith hemophilia A.

Hemophilia A is an inherited bleeding disorder that results in frequentspontaneous and traumatic bleeding into the joints and soft tissues.Mannucci P M, Tuddenham E G D, N Engl J Med, 344:1773-1779 (2001). Wheninadequately treated, this bleeding leads to chronic arthropathy,disability, and increased risk of death. Soucie J M et al., Blood.96(2):437-442 (2000).

Plasma-derived FVIII (pdFVIII) and recombinant human FVIII (rFVIII)products are utilized for treatment (on-demand therapy) and prevention(prophylaxis therapy) of bleeding episodes. rFVIII was developed toreduce the risk of blood-borne pathogen transmission following thewidespread contamination of plasma products with HIV and hepatitisviruses, and to secure an adequate supply of FVIII product. However,hemostatic protection with current FVIII products is temporally limiteddue to a short half-life (t_(1/2)) of approximately 8-12 hours,requiring prophylactic injections three times per week or every otherday for most patients in order to maintain FVIII levels above 1%, alevel that has been established as protective against most spontaneousbleeding episodes. Manco-Johnson et al., New Engl J Med. 357(6):535-44(2007).

Many studies have shown that, even at high doses, on-demand therapy isnot effective in preventing arthropathy. Aledort L. et al., J InternMed. 236:391-399 (1994); Petrini P. et al., Am J Pediatr Hematol Oncol.13:280-287 (1991). The benefits of prophylactic therapy have beendemonstrated in numerous clinical studies^(4,6-15) and Manco-Johnson etal., supra, established that children started on primary prophylaxisafter their first joint bleed had significantly fewer bleeds and lessjoint damage than children treated on-demand.

Compared to on-demand treatment, prophylactic therapy also decreasesdisability, hospitalization rate, and time lost from school orwork;^(6,16) and improves quality of life for patients and theirfamilies.¹⁷ However, prophylactic therapy often requires use of centralvenous access devices in children, and their attendant risks ofinfection, sepsis, and thrombosis. In addition, despite the benefits,acceptance of and compliance with prophylaxis decreases with age, inpart because of inconvenience and invasiveness.^(18,19) Thus, a rFVIIIproduct with a prolonged plasma t_(1/2) would potentially be of benefit.Lillicrap D., Current Opinion in Hematology 17:393-397 (2010).

rFVIIIFc is a recombinant fusion protein composed of a single moleculeof B-domain deleted rFVIII covalently linked to the human IgG₁ Fcdomain. Potential advantages of Fc-fusion proteins include bettertolerability and prolonged hemostatic protection, and the Fc domainrepresents a natural molecule with no known inherent toxicity.^(21,22)Attachment to the IgG₁ Fc domain permits binding to the neonatal Fcreceptor (FcRn), which is expressed in many cell types, includingendothelial cells. FcRn expression remains stable throughout life and isresponsible for protecting IgG₁ and Fc-fusion proteins from lysosomaldegradation, thus prolonging the t_(1/2) of the protein.^(21,23)Numerous proteins within the circulation are internalized into the cellslining the vasculature via nonspecific pinocytosis and are trafficked toendosomal and lysosomal degradation pathways.

Fc proteins interact with FcRn, resident within endosomes. Endosomescontaining FcRn direct the Fc fusion proteins back to the plasmamembrane, releasing them into circulation in a pH-dependent manner,²⁴thereby avoiding lysosomal degradation. This recycling approach has beenused successfully to extend the t_(1/2) of therapeutic biologics; anumber of Fc fusion-based drugs have been approved for clinical use (egetanercept, romiplostim) and others are in development.^(25,26)

Preclinical data for rFVIIIFc indicate that FVIII can be rescued fromdegradation by a natural protective pathway mediated by FcRn, thusextending t_(1/2). In Hemophilia A mice and dogs, terminal plasmat_(1/2) for rFVIIIFc was approximately 2 times longer than withrFVIII.^(27,28) Based on these data, we conducted a first-in-humanclinical study to investigate the safety and PK of a long-lastingrFVIIIFc fusion protein in subjects with hemophilia A.

Study Design: This open-label, dose-escalation, multicenter Phase 1/2astudy in previously treated patients with severe hemophilia Ainvestigated the safety of rFVIIIFc and its pharmacokinetics (PK)compared with ADVATE® (antihemophilic factor [recombinant],plasma/albumin-free method, octocog alfa, Baxter Healthcare). This studywas performed in accordance with the US CFR and ICH Guidelines on GoodClinical Practices. Prior to any testing, approval from participatingInstitutional Review Boards and written informed consents from allsubjects were obtained. The study design was sequential; a single doseof ADVATE® was administered at 25 or 65 IU/kg followed by an equal doseof rFVIIIFc (FIG. 8). Both drugs were injected intravenously overapproximately 10 minutes. The two dose levels were expected to bracketthe typical therapeutic dose ranges. Subjects were followed for 28 daysafter receiving rFVIIIFc for safety analyses, including testing foranti-FVIII antibodies and inhibitors at 14 and 28 days post-injection.Plasma FVIII activity was measured in subjects before injection, 10 and30 minutes, 1, 3, 6, 9, 24, 48, 72, 96, 120, and 168 hours (7 days)after rFVIIIFc injection, with additional samples at 192, 216, and 240hours (10 days) for subjects dosed at 65 IU/kg of rFVIIIFc. Plasma FVIIIactivity was measured at the same time points after ADVATE® treatment,through 72 hours for the 25 IU/kg group and 96 hours for the 65 IU/kggroup.

Subjects: Male subjects were at least 12 years of age with severehemophilia A (defined as FVIII activity level <1%) and had at least 100documented prior exposure days to FVIII concentrates (pdFVIII orrFVIII). Subjects with known hypersensitivity to mouse or hamsterprotein, history of inhibitor or detectable inhibitor titer atscreening, or who were taking any medications that could affecthemostasis or systemic immunosuppressive drugs, or who experienced anactive bacterial or viral infection (other than hepatitis or HIV) within30 days of screening were excluded. Subject's genotype was recorded atstudy entry, when known.

Treatment Product: The human rFVIIIFc and Fc transgenes were stablytransfected into HEK293 cells and the cell line was extensively testedfor stability, sterility, and viral contamination to ensure safety. Thepurified drug product is composed of a monomeric B-domain-deleted FVIIIcovalently linked through its carboxy-terminus to the N-terminus of anFc monomer, which forms a disulfide bond with a second Fc monomer duringsynthesis and secretion from the cells. rFVIIIFc was purified bychromatography and nanofiltration, and was fully active in one-stage andchromogenic clotting assays relative to commercially available rFVIIIpreparations. It was supplied as a frozen liquid containing 1000 IU per2 mL of solution and formulated with L-histidine (pH 7), sodiumchloride, calcium chloride, sucrose, mannitol, and Polysorbate 20. Forinjection, the product was diluted with saline solution (0.9% NaCl).

Outcome Measures: The primary objective of the study was safety,evaluated through physical examination, reporting of treatment-emergentadverse events (AEs), development of antibodies, and laboratorymonitoring over time. The secondary objectives included parametersderived from PK analyses. Laboratory assessments included prothrombintime, activated partial thromboplastin time (aPTT), internationalnormalized ratio, levels of D-dimer, von Willebrand factor (vWF)antigen, standard hematology and blood chemistry tests, and urinalysis.

FVIII activity was measured by the one-stage clotting (aPTT) assay on aSiemens BCS-XP analyzer using commercial reagents (Dade Actin FSL) withcalibration against a normal reference plasma (Precision BiologicsCRYOcheck™) traceable to the World Health Organization (WHO) 5^(th)International Standard (IS) for human plasma. In addition to the aPTTassay, FVIII activity was measured by a chromogenic substrate assay²⁹using a commercially available kit (Aniara BIOPHEN FVIII:C) thatcomplies with European Pharmacopoeia recommendations. The chromogenicassay was calibrated against normal human reference plasma(Instrumentation Laboratories ORKE45), which also had a potency assignedagainst the WHO 5th IS human plasma standard.

The lower limit of quantification (LLOQ) for the one-stage andchromogenic assays was 0.5 IU/dL and 0.4 IU/dL, respectively. FVIIIinhibitors were measured by the—*Nijmegen-modified Bethesda assay andless than 0.6 BU/mL was considered negative. Anti-rFVIIIFc antibodieswere assessed using a specific bridging electrochemiluminescentimmunoassay which uses biotin and sulfo-tagged rFVIIIFc. Assaysensitivity was determined to be 89 ng/mL using an anti-human FVIIImonoclonal antibody as a surrogate control. Exploratory whole bloodrotation thromboelastometry (ROTEM®) was performed in two subjects, onefrom each dose level, at various time points to assess the improvementin global hemostasis following injection with ADVATE® and rFVIIIFc.

Pharmacokinetic Analyses: A user-defined one-compartment dispositionmodel, which automatically estimates the endogenous FVIII level andsubsequent residual decay, was utilized in WinNonLin for analysis of theindividual subject plasma FVIII activity-versus-time data following asingle administration of ADVATE® or rFVIIIFc. Actual sampling times,doses, and duration of injection were used for calculations ofparameters including maximum activity (Cmax), t_(1/2), clearance (CL),volume of distribution at steady-state (V_(ss)), area under the curve(time zero extrapolated to infinity [AUC_(INF)]), mean residence time(MRT), and incremental recovery.

Monte Carlo Simulation of rFVIIIFc Activity-Versus-Time Profile—Toconstruct FVIII activity-time profiles following dosing regimens of 25IU/kg or 65 IU/kg, a Monte Carlo simulation was conducted using thepopulation PK model of ADVATE® and rFVIIIFc. The mean estimates of modelparameters (CL, volume of distribution) in the tested population, theinter-individual variance, and the residual variability were estimatedbased on the one-stage (aPTT) clotting assay activity data of ADVATE®and rFVIIIFc from 16 subjects in this Phase1/2a study. Five hundredsubjects were simulated with 15 sampling points for each subject foreach dosing regimen. The percentage of the population with FVIIIactivity above or equal to 1% and 3% at different times followingdifferent dosing regimens of ADVATE® or rFVIIIFc was estimated.

Statistical Analyses—Selected PK parameters for rFVIIIFc and ADVATE®were compared using an analysis of variance model. PK parameters werelog-transformed for these analyses and estimated means, meandifferences, and confidence intervals on the log-scale were transformedto obtain estimates for geometric means, geometric mean ratios (GMR),and confidence intervals, respectively, on the original scale. The GMRis the geometric mean of the intra-subject ratio of the rFVIIIFc PKparameter value to the ADVATE® PK parameter value.

Results

Subject Disposition—Nineteen subjects were enrolled in the study; 16underwent PK evaluation for both ADVATE® and rFVIIIFc. One subjectself-administered his previous product prior to completing the wash-outperiod following the dose with ADVATE® and was thus excluded from the PKanalysis, but was included in the safety analysis. Three subjects werediscontinued from the study before receiving either study drug: onevoluntarily withdrew; a second was withdrawn by the Investigator fornon-compliance; and one was withdrawn at the Sponsor's request due tocompletion of study enrollment. Of the subjects dosed, six subjectsreceived 25 IU/kg and 10 subjects received 65 IU/kg of both ADVATE® andrFVIIIFc. Mean age was 40.3 years (23 to 61 years). Genotypicidentification was collected for seven subjects; inversion of intron 22was reported in six subjects; and a frame-shift defect was reported inone subject. The genotype was unknown for nine subjects. Thirteensubjects had hepatitis C antibodies, four of whom were also positive forHIV.

Safety-Forty-four treatment-emergent AEs were reported by 11 (69%)subjects during the treatment and follow-up periods. This included theday of dosing with Advate or rFVIIIFc through a 28-day post-dosingobservation period. The majority of events were considered mild and noneled to withdrawal from the study. One event, dysgeusia, occurredtransiently in one subject while receiving a 65 IU/kg dose of rFVIIIFcand was considered related to rFVIIIFc.

One subject experienced an anxiety attack after receiving 65 IU/kg ofrFVIIIFc which resulted in 21 AEs, 19 of which were graded as mild, andtwo of which (headache and photophobia) were rated as moderate. Neitherwas deemed related to rFVIIIFc by the Investigator.

No serious bleeding episodes were reported. No evidence of allergicreactions to injection was detected. All plasma samples tested negativefor FVIII inhibitors and anti-rFVIIIFc antibodies. No signs of injectionsite reactions were observed. No clinically meaningful changes inabnormal laboratory values were reported.

Pharmacokinetics: Correlation Between aPTT and Chromogenic Activity forrFVIIIFc in Plasma—ADVATE® and rFVIIIFc activities were determined inthe same assays using commercially available reagents and calibrationagainst normal human plasma standards. There was a strong correlationbetween the results obtained by the one-stage clotting assay and thechromogenic assay in samples that had an activity above the LLOQ.Correlation coefficients (Pearson R²) of 0.94 and 0.95 were observedbetween the two assay results for 151 samples following ADVATE® dosingand 185 samples following rFVIIIFc dosing, respectively. Compared to theaPTT results, the chromogenic FVIII activities were, on average, 21%higher for ADVATE® and 32% higher for rFVIIIFc, not statisticallysignificant (FIG. 9). This observation led to a slightly higherestimation of exposure parameters by the chromogenic assessment for bothdrugs. The apparent higher FVIII recoveries by the chromogenic assay aretypical for recombinant FVIII products tested in clinical assays, andare in agreement with most other marketed FVIII products.³⁰⁻³²

Improved Pharmacokinetics for rFVIIIFc—The primary PK estimates werederived from one-stage (aPTT) clotting assay activity data. In subjectswho received 25 or 65 IU/kg of ADVATE® followed by an equal dose ofrFVIIIFc, the plasma FVIII activity rose sharply and reached C_(max)within the first hour following dosing. The subsequent decline of theobserved FVIII activity exhibited monoexponential decay characteristicsuntil the baseline FVIII activity was reached (FIG. 10). The C_(max)increased proportionally to the dose, but was comparable between equaldoses of ADVATE® and rFVIIIFc (Table 8) The total exposure (AUC_(INF))also increased proportionally to the dose. However, the AUC_(INF) ofrFVIIIFc was 1.48 and 1.56-fold greater than that of ADVATE® at 25 IU/kg(p=0.002) and 65 IU/kg (p<0.001), respectively (Table 8).

TABLE 8 PK Parameters by One-Stage (aPTT) Assay for rFVIIIFc andADVATE ® Per Dose Group Dose: 25 IU/kg (N = 6) Dose: 65 IU/kg (N = 9)Geom. Mean Geom. Mean ADVATE ® rFVIIIFc Ratio ADVATE ® rFVIIIFc RatioGeom. Mean Geom. Mean [95% CI] Geom. Mean Geom. Mean [95% CI] Parameter[95% CI] [95% CI] (p-value) [95% CI] [95% CI] (p-value) C_(max)_OBS 63.660.5 0.952 133 119 0.895 (IU/dL) [59.1, 68.3] [53.1, 69.0] [0.819, 1.11][105, 168] [103, 136] [0.795, 1.01] (p = 0.440) (p = 0.061) AUC_(INF)994 1480 1.48 1800 2800 1.56 (hr*IU/dL) [723, 1370] [1160, 1880] [1.26,1.76] [1350, 2400] [1980, 3970] [1.33, 1.83] (p = 0.002) (p < 0.001)t_(1/2) (hr) 12.2 18.8 1.54 11.0 18.8 1.70 [9.14, 16.3] [14.8, 23.8][1.40, 1.69] [8.76, 13.9] [14.3, 24.5] [1.54, 1.89] (p < 0.001) (p <0.001) MRT (hr) 17.5 27.0 1.54 15.8 27.0 1.71 [13.1, 23.4] [21.3, 34.2][1.40, 1.69] [12.6, 19.9] [20.6, 35.3] [1.54, 1.89] (p < 0.001) (p <0.001) CL 2.49 1.68 0.673 3.61 2.32 0.642 (mL/hour/kg) [1.80, 3.45][1.31, 2.15] [0.569, 0.796] [2.71, 4.83] [1.64, 3.29] [0.547, 0.753] (p= 0.002) (p < 0.001) V_(ss) 43.9 45.4 1.04 57.4 62.8 1.09 (mL/kg) [39.3,49.0] [39.3, 52.5] [0.947, 1.13] [48.3, 68.3] [55.2, 71.5] [0.976, 1.22](p = 0.357) (p = 0.107) Incremental 2.56 2.44 0.952 2.04 1.83 0.894Recovery [2.36, 2.78] [2.12, 2.81] [0.819, 1.11] [1.61, 2.59] [1.59,2.10] [0.795, 1.01] (IU/dL per (p = 0.444) (p = 0.060) IU/kg) CI =Confidence Interval; Geom. Mean = Geometric Mean; OBS = observed.Estimated means, 95% CI for means, and mean differences were transformedto obtain estimated geometric means, 95% CI for geometric means, andgeometric mean ratios, respectively.

The t_(1/2), MRT, CL, and V_(ss) appeared to be independent of dose(Table 8). The geometric mean t_(1/2) of rFVIIIFc was 18.8 hours forboth the 25 IU/kg and 65 IU/kg dose groups. This represents a 1.54 and1.70-fold improvement over that of ADVATE® (12.2 hours and 11.0 hours)at equivalent doses (p<0.001), respectively (Table 8). The sameintra-subject improvement was observed in the MRT of rFVIIIFc (27.0hours for both dose groups) compared with ADVATE® (17.5 hours for the 25IU/kg and 15.8 hours for the 65 IU/kg) (p<0.001). Consistent withimprovement in the t_(1/2) and MRT was a corresponding 1.49 and1.56-fold reduction in intra-subject CL at doses of 25 IU/kg (p=0.002)and 65 IU/kg (p<0.001), respectively. There were no significantdifferences in V_(ss) and incremental recovery between ADVATE® andrFVIIIFc. Therefore, within each subject, rFVIIIFc demonstrated animproved PK profile compared with ADVATE®. It was also observed that thepatients with shorter half-life on ADVATE® had shorter half-life onrFVIIIFc, and patients with longer half-life on ADVATE® had longerhalf-life on rFVIIIFc.

The improved PK profile of rFVIIIFc resulted in increased timepost-dosing to 1% FVIII activity which was 1.53 and 1.68-fold longerrespectively, than with ADVATE® at 25 IU/kg (p<0.001) and 65 IU/kg(p<0.001) (data not shown), suggesting a potentially longer therapeuticduration for rFVIIIFc.

The favorable PK profile of rFVIIIFc relative to ADVATE® was alsodemonstrated by FVIII activity measured in the chromogenic assay (Table9), which was comparable to data derived from aPTT assays. Theestimation of exposure, ie, C_(max) and AUC_(INF), was slightly higher,however, based on the chromogenic assay than on the one-stage (aPTT)clotting assay for both ADVATE® and rFVIIIFc.

TABLE 9 PK Parameters by Two-Stage (Chromogenic) Assay for rFVIIIFc andADVATE ® Per Dose Group Dose: 25 IU/kg (N = 6) Dose: 65 IU/kg (N = 9)Geom. Mean Geom. Mean ADVATE ® rFVIIIFc Ratio ADVATE ® rFVIIIFc RatioGeom. Mean Geom. Mean [95% CI] Geom. Mean Geom. Mean [95% CI] Parameter[95% CI] [95% CI] (p-value) [95% CI] [95% CI] (p-value) C_(max)_OBS 75.576.5 1.01 175 182 1.04 (IU/dL) [65.5, 87.1] [64.9, 90.1] [0.940, 1.09][143, 215] [146, 227] [0.900, 1.20] (p = 0.686) (p = 0.571) AUC_(INF)1060 1660 1.57 2270 4280 1.89 (hr*IU/dL) [822, 1360] [ 1300, 2120][1.38, 1.80] [1670, 3070] [2960, 6190] [1.61, 2.21] (p < 0.001) (p <0.001) t_(1/2) (hr) 10.5 16.7 1.59 10.8 19.8 1.84 [8.49, 12.9] [13.8,20.1] [1.35, 1.87] [8.16, 14.2] [14.3, 27.5] [1.60, 2.12] (p < 0.001) (p< 0.001) MRT (hr) 15.0 23.9 1.59 15.4 28.5 1.85 [12.2, 18.6] [19.8,28.9] [1.35, 1.87] [11.7, 20.4] [20.5, 39.6] [1.61, 2.12] (p < 0.001) (p< 0.001) CL 2.35 1.49 0.636 2.87 1.52 0.530 (mL/hour/kg) [1.80, 3.06][1.16, 1.92] [0.557, 0.727] [2.12, 3.89] [1.05, 2.20] [0.453, 0.620] (p< 0.001) (p < 0.001) Vss 35.5 35.9 1.01 44.5 43.4 0.975 (mL/kg) [30.5,41.3] [30.4, 42.3] [0.898, 1.14] [36.7, 54.1] [38.2, 49.4] [0.863, 1.10](p = 0.822) (p = 0.653) Incremental 3.05 3.09 1.01 2.70 2.80 1.04Recovery [2.62, 3.54] [2.61, 3.66] [0.940, 1.09] [2.20, 3.31] [2.24,3.50] [0.900, 1.20] (IU/dL per (p = 0.679) (p = 0.571) IU/kg) CI =Confidence Interval; Geom. Mean = Geometric Mean; OBS = observed.Estimated means, 95% CI for means, and mean differences were transformedto obtain estimated geometric means, 95% CI for geometric means, andgeometric mean ratios, respectively.

Correlation Between von Willebrand Factor and Disposition of rFVIIIFc—

Because the majority of FVIII in circulation is in complex with VWF³³and because the genome-wide association study has identified that thegenetic determinants of FVIII levels are primarily dependent on VWFlevels,³⁴ we examined the association between VWF and rFVIIIFc. A strongcorrelation was observed between VWF levels and CL and t_(1/2) for bothrFVIIIFc and ADVATE®. As shown in FIG. 10, as the level of VWFincreased, the CL of rFVIIIFc (p=0.0016) and of ADVATE® (p=0.0012)decreased.

The opposite relationship was observed between the level of VWF andt_(1/2). As the level of VWF increased, the t_(1/2) of rFVIIIFc(p=0.0003) and of ADVATE® (p<0.0001) increased. This correlationsuggests that the Fc moiety of rFVIIIFc does not alter the role of VWFin protecting FVIII from clearance.

Effects of Prolonged PK of rFVIIIFc on Whole Blood ROTEM®—Prior toadministration of study drug, blood from one subject in each dose groupwas spiked with an equal dose of rFVIIIFc or ADVATE® and analyzed bywhole blood ROTEM®. Clotting time (CT) was linear with respect to thedose in the range of approximately 1% to 100% of normal, and the doseresponse was comparable between rFVIIIFc and ADVATE® in the same subject(data not shown), indicating comparable potency of rFVIIIFc and ADVATE®in clot formation.

Despite the variable baseline CT due to residual FVIII levels prior tothe administration of ADVATE® or rFVIIIFc, both products effectivelycorrected the CT to comparable levels 30 minutes post-dosing (FIG. 12).The improvement in CT was better sustained by rFVIIIFc than ADVATE®after 3 hours following a dose of 25 IU/kg (FIG. 12A), and after 24hours following a dose of 65 IU/kg (FIG. 12B).

rFVIIIFc was well tolerated by subjects at both doses. There were noclinically significant changes observed in hematology, blood chemistry,or urinalysis parameters. The majority of AEs were mild, unrelated torFVIIIFc, and resolved without sequelae. No serious AEs or deathsoccurred during the study, and no subjects at either dose developedneutralizing or binding antibodies to rFVIIIFc.

rFVIIIFc demonstrated a significantly improved FVIII activity PK profilerelative to ADVATE®, with t_(1/2) and MRT across dose levels being 1.54to 1.71-fold longer, as measured by the one-stage (aPTT) clotting assayand 1.59 to 1.84-fold longer by the two-stage chromogenic assay. Theprolonged activity of rFVIIIFc predicts possible prolonged efficacy,allowing for a less frequent dosing regimen in the prophylactictreatment of patients with Hemophilia A.

Adopting the PK parameters derived from this study, the Monte Carlosimulation predicts that a higher percentage of patients receivingrFVIIIFc will sustain FVIII levels above 1% or 3% as compared withpatients receiving equal doses of ADVATE® (Table 10). For example, at adose of 25 IU/kg, 12.2% of ADVATE® patients versus 71.2% of rFVIIIFcpatients are predicted to have FVIII trough levels above 1% on Day 4; ata dose of 65 IU/kg, 11.0% of ADVATE® patients versus 66.4% of rFVIIIFcpatients are predicted to have FVIII levels above 3% on Day 4. Clinicaltrials in larger numbers of patients are planned to confirm results fromthis Phase 1/2a study and from the Monte Carlo simulation predictions.

TABLE 10 Predicted Percentage of Subjects Achieving FVIII Trough LevelsAbove 1% and 3% of Normal at a Specified Dose Regimen of ADVATE ® orrFVIIIFc Timepoint following ADVATE ® rFVIIIFc dosing (Day) 25 IU/kg 65IU/kg 25 IU/kg 65 IU/kg Percent of Subjects with FVIII Trough Levelsabove 1% 3 40.0 67.8 92.6 99.0 4 12.2 31.0 71.2 90.0 5 4.20 13.6 39.471.6 7 0.200 1.40 7.80 26.4 Percent of Subjects with FVIII Trough Levelsabove 3% 3 10.6 34.6 62.2 91.0 4 1.60 11.0 25.4 66.4 5 0.200 3.20 7.0036.2 7 0 0.200 0.400 6.60

Despite the success of Fc fusion technology in prolonging circulatingt_(1/2) for a variety of protein therapeutics, rFVIII was considered toolarge to successfully produce dimeric Fc fusions. We thus created amonomeric Fc fusion protein whereby a single effector molecule wascovalent linked to a dimeric Fc, enabling binding to intracellular FcRnand subsequent recycling.^(21,22) In vitro coagulation assaysdemonstrate no loss of specific activity for rFVIIIFc, compared toB-domain deleted or native FVIII, by either clotting or chromogenicassays, using commercially available reagents and commonly used FVIIIreference standards (JAD, TL, SCL, et al., manuscript submitted August,2011). In addition, these results indicate that rFVIIIFc can be reliablyassayed in a clinic setting by either the one-stage assay or thechromogenic method.

In summary, this Phase 1/2a clinical trial is the first trial todemonstrate the safety and prolonged t_(1/2) of rFVIIIFc in patientswith severe hemophilia A. A pivotal Phase 3 study is ongoing withrFVIIIFc to establish effective prophylaxis dosing regimens forindividuals with hemophilia A.

Example 11

A novel single-chain (SC) isoform of factor VIII (FVIII), resulting fromincomplete proteolysis at residue R1648 during biosynthesis, may providesuperior manufacturability and stability relative to native FVIII. Asingle recombinant B domain deleted factor VIII molecule fused to animmunoglobulin Fc domain (rFVIIIFc) and its purified SC counterpart(SC-rFVIIIFc) exhibited similar specific activity in one stage clottingassays using plasma depleted of von Willebrand factor (VWF), butSC-rFVIIIFc exhibited lower specific activity in the presence of VWF.This study was undertaken to determine if VWF-bound rFVIIIFc,SC-rFVIIIFc and rBDD-FVIII (XYNTHA®, REFACTO AF®) differ with respect tothrombin-mediated proteolytic release from VWF.

Equimolar amounts of rFVIIIFc, SC-rFVIIIFc, and rBDD-FVIII were capturedon an optical biosensor chip on which human VWF had been immobilized byamine coupling. Human α-thrombin at a range of concentrations wasinfused over the chip surface, and the rates of FVIII release fromimmobilized VWF were monitored in real time. The half maximal effectiveconcentration (EC₅₀) of α-thrombin was determined for each FVIIIspecies.

α-thrombin EC₅₀ values for rFVIIIFc and rBDD-FVIII were comparable(3.7±0.2 U/mL and 3.2±0.3 U/mL, respectively), whereas the EC₅₀ valuefor SC-rFVIIIFc was greater than 3-fold higher (11.7±0.9 U/mL). Thisfinding that SC-rFVIIIFc is released more slowly from VWF than areeither rFVIIIFc or rBDD-FVIII is consistent with a previously observedfinding regarding the activities of rFVIIIFc and SC-rFVIIIFc in aone-stage clotting assay (aPTT) in which SC-FVIIIFc had a lower apparentactivity only when VWF was present in the assay plasma sample. However,all samples possessed equivalent activities in a mouse bleeding model,indicating that responsiveness of FVIII preparations to thrombin in therelease of FVIII from VWF does not correlate with efficacy in vivo.

TABLE1 Polynucleotide Sequences A. B-Domain Deleted FVIIIFc(i) B-Domain Deleted FVIIIFc Chain DNA Sequence (FVIII signal peptideunderlined, Fc region in bold) (SEQ ID NO: 1, which encodes SEQ ID NO: 2)661                                A TGCAAATAGA GCTCTCCACC TGCTTCTTTC721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT1501 CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA3721 CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT4201 GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA4381 GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT4441 TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT4681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC4741 CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC4801 AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC4861 AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA4921 ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC4981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT5041 GCGAGGCACA GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC5101 TCCTGGGCGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT5161 CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA5221 AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG5281 AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC5341 TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA5401 AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT5461 CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC5521 CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA5581 CGCCTCCCGT GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA5641 AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA5701 ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A(ii) Fc DNA sequence (mouse Igκsignal peptide underlined) (SEQ ID NO: 3, which encodes SEQ ID NO: 4)7981                                                  ATGGA GACAGACACA8041 CTCCTGCTAT GGGTACTGCT GCTCTGGGTT CCAGGTTCCA CTGGTGACAA AACTCACACA8101 TGCCCACCGT GCCCAGCACC TGAACTCCTG GGAGGACCGT CAGTCTTCCT CTTCCCCCCA8161 AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC8221 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT8281 AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC8341 CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC8401 AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA8461 CCACAGGTGT ACACCCTGCC CCCATCCCGC GATGAGCTGA CCAAGAACCA GGTCAGCCTG8521 ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG8581 CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGTTGG ACTCCGACGG CTCCTTCTTC8641 CTCTACAGCA AGCTCACCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC8701 TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG8761 GGTAAA B. Full Length FVIIIFc(i) Full Length FVIIIFc DNA Sequence (FVIII signal peptide underlined, Fcregion in bold) (SEQ ID NO: 5, which encodes SEQ ID NO: 6) 661                                        ATG CAAATAGAGC TCTCCACCTG 721CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC 781AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG 841ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC 901TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT 961GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA 1021GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC 1081TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT 1141CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC 1201TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA 1261TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC 1321ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA 1381CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC 1441TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA 1501CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT 1561ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TGGAAATCTC- 1621GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT 1681TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG 1741TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA 1801TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT 1861CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA 1921AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG 1981TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT 2041GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT 2101GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC 2161AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG 2221GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT 2281CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT 2341GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG 2401CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA 2461CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA 2521GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA 2581AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT 2641TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT 2701TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC 2761CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT 2821TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC 2881TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA 2941CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC 3001TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC 3061TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA 3121TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA 3181AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA 3241CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT 3301TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGGGGACAA CTGCAGCAAC 3361AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT 3421TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT 3481GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT 3541TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA 3601ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG 3661TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC 3721CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC 3781TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG 3841GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG 3901AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC 3961TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC 4021ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT 4081ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT 4141AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA 4201AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC 4261AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA 4321TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT 4381AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT 4441ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA 4501AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC 4561AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA 4621GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA 4681ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA 4741AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC 4801GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC 4861CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC 4921CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT 4981CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA 5041AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC 5101CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC 5161AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC 5221TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA 5281AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC 5341AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT 5401AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA 5461CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA 5521AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC 5581AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA 5641AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA 5701AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC 5761AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG 5821CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA 5881TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA 5941GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG 6001TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA 6061TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT 6121TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC 6181TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA 6241GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC 6301AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG 6361ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG 6421GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA 6481TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT 6541ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG 6601CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA 6661GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT 6721ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG 6781GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC 6841TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT GGGCCCCAAA 6901GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC 6961TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA CCCAGGGTGC 7021CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG 7081GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA 7141TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT 7201CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG 7261TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA 7321GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG 7381ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG 7441GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA 7501ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA 7561TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC 7621CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA 7681CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA 7741GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC TCCTGGGCGG 7801ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC 7861TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG 7921GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA 7981CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA 8041GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 8101CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA 8161GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT 8221CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT 8281GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG 8341GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 8401GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A(ii) Fc (same sequence as A(ii)(SEQ ID NO: 3))]

TABLE 2 Polypeptide SequencesA. B-Domain Deleted FVIII-Fc Monomer Hybrid (BDD FVIIIFc monomer dimer):created by coexpressing BDD FVIIIFc and Fc chains. Construct =HC-LC-Fc fusion. An Fc expression cassette is cotransfectedwith BDDFVIII-Fc to generate the BDD FVIIIFc monomer-. For the BDD FVIIIFcchain, the Fc sequence is shown in bold; HC sequence is shown in doubleunderline; remaining B domain sequence is shown in italics. Signal peptidesare underlined.i) B domain deleted FVIII-Fc chain (19 amino acid signal sequence underlined)(SEQ ID NO: 2) MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPR SFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKii) Fc chain (20 amino acid heterologous signal peptide from mouse Igκchain underlined)(SEQ ID NO: 4) METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKB. Full length FVIIIFc monomer hybrid (Full length FVIIIFc monomer dimer):created by coexpressing FVIIIFc and Fc chains. Construct =HC-B-LC-Fc fusion. An Fc expression cassette is cotransfectedwith full length FVIII-Fc to generate the full length FVIIIFc monomer. Forthe FVIIIFc chain, the Fc sequence is shown in bold; HC sequence is shown indouble underline; B domain sequence is shown in italics. Signal peptides areunderlined.i) Full length FVIIIFc chain (FVIII signal peptide underlined (SEQ ID NO: 6)MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPR SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKii) Fc chain (20 amino acid heterologous signal peptide from mouse Igκchain underlined) (SEQ ID NO: 4) METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

CITED REFERENCES

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1-264. (canceled)
 265. A pharmaceutical composition comprising (i) achimeric polypeptide, which comprises a Factor VIII (FVIII) portion anda second portion, and (ii) at least one pharmaceutically acceptableexcipient, wherein at least about 1%, about 5%, about 10%, about 15%,about 20%, or about 25% of the chimeric polypeptide comprises singlechain FVIII in the FVIII portion.
 266. The pharmaceutical composition ofclaim 265, wherein about 1%-about 10%, about 5%-about 15%, about10%-about 20%, about 15%-about 25%, about 20%-about 30%, about 25%-about35%, or about 30%-about 40% of the chimeric polypeptide is single chainFVIII in the FVIII portion.
 267. The pharmaceutical composition of claim265, wherein at least 75% of the chimeric polypeptide comprisesprocessed FVIII in the FVIII portion.
 268. The pharmaceuticalcomposition of claim 265, wherein about 15% to about 25% of the chimericpolypeptide comprises single chain FVIII in the FVIII portion and about75% to about 85% of the chimeric polypeptide comprises processed FVIIIin the FVIII portion.
 269. The pharmaceutical composition of claim 265,(i) wherein about 25% of the chimeric polypeptide comprises single chainFVIII in the FVIII portion and about 75% of the chimeric polypeptidecomprises processed FVIII in the FVIII portion, (ii) wherein about 20%of the chimeric polypeptide comprises single chain FVIII in the FVIIIportion and about 80% of the chimeric polypeptide comprises processedFVIII in the FVIII portion, or (iii) wherein about 15% of the chimericpolypeptide comprises single chain FVIII in the FVIII portion and about85% of the chimeric polypeptide comprises processed FVIII in the FVIIIportion.
 270. The pharmaceutical composition of claim 265, wherein thesingle chain FVIII comprises a heavy chain and a light chain connectedby a peptide bond.
 271. The pharmaceutical composition of claim 267,wherein the processed FVIII comprises two chains, a first chaincomprising a heavy chain of FVIII and a second chain comprising a lightchain of FVIII, wherein the first chain and the second chain areassociated by a metal bond.
 272. The pharmaceutical composition of claim265, wherein the chimeric polypeptide is a long-acting FVIIIpolypeptide.
 273. The pharmaceutical composition of claim 265, whereinthe second portion comprises an Fc region, albumin, a PAS sequence,transferrin, CTP (28 amino acid C-terminal peptide (CTP) of hCG with its4 O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES),albumin binding polypeptide, albumin-binding small molecules, or two ormore combinations thereof.
 274. The pharmaceutical composition of claim265, wherein the second portion comprises an Fc region.
 275. Thepharmaceutical composition of claim 265, wherein the FVIII portioncomprises B-domain deleted FVIII or full-length, mature FVIII.
 276. Thepharmaceutical composition of claim 265, wherein residue 1645corresponding to full-length, mature FVIII, residue 1648 correspondingto full-length, mature FVIII, or both residues in the single chain FVIIIis arginine.
 277. The pharmaceutical composition of claim 265, whereinthe chimeric polypeptide has a half-life longer than a polypeptideconsisting of the FVIII portion.
 278. The pharmaceutical composition ofclaim 265, which is more stable than a composition comprising processedFVIII without single chain FVIII.
 279. The pharmaceutical composition ofclaim 265, which is suitable for administration to a human.
 280. Thepharmaceutical composition of claim 265, which is suitable forsubcutaneous, intradermal, intravascular, intravenous, intramuscular,spinal, intracranial, intrathecal, intraocular, periocular,intraorbital, intrasynovial or intraperitoneal injection to a human.281. The pharmaceutical composition of claim 265, which is lyophilized.282. A method of preventing, decreasing, or treating a bleeding episodein a subject comprising administering to the subject an effective amountof the pharmaceutical composition of claim
 265. 283. A method ofprophylactic treatment of a bleeding episode in a subject comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 265. 284. A method of on-demand treatment of ableeding episode in a subject comprising administering to the subject aneffective amount of the pharmaceutical composition of claim
 265. 285. Amethod of tailored prophylactic treatment of a bleeding episode in asubject comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim
 265. 286. The method of claim282, wherein the bleeding episode is associated with a disease orcondition comprising hemarthrosis, muscle bleed, oral bleed, hemorrhage,hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis (headtrauma), gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, or bleeding in the illiopsoassheath.
 287. The method of claim 283, wherein the bleeding episode isassociated with a disease or condition comprising hemarthrosis, musclebleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,trauma, trauma capitis (head trauma), gastrointestinal bleeding,intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracichemorrhage, bone fracture, central nervous system bleeding, bleeding inthe retropharyngeal space, bleeding in the retroperitoneal space, orbleeding in the illiopsoas sheath.
 288. A method of maintaininghomeostatis in a subject in need of a surgery comprising administeringto the subject an effective amount of the pharmaceutical composition ofclaim
 265. 289. A method of perioperative management of a subjectbefore, after, or during a surgery comprising administering to thesubject an effective amount of the pharmaceutical composition of claim265.
 290. The method of claim 289, wherein the surgery is minor surgery,major surgery, tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, total knee replacement, craniotomy, osteosynthesis, traumasurgery, intracranial surgery, intra-abdominal surgery, intrathoracicsurgery, or joint replacement surgery.
 291. The method of claim 282,wherein the effective amount of the pharmaceutical composition is 10IU/kg-20 IU/kg, 20 IU/kg-30 IU/kg, 30 IU/kg-40 IU/kg, 40 IU/kg-50 IU/kg,50 IU/kg-60 IU/kg, 60 IU/kg-70 IU/kg, 70 IU/kg-80 IU/kg, 80 IU/kg-90IU/kg, 90 IU/kg-100 IU/kg, 100 IU/kg-110 IU/kg, 110 IU/kg-120 IU/kg, 120IU/kg-130 IU/kg, 130 IU/kg-140 IU/kg, or 140 IU/kg-150 IU/kg.
 292. Themethod of claim 282, wherein a dosing interval of the pharmaceuticalcomposition is once every 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, or24-168 hours or longer.